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Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545,
DOI 10.1007/978-1-4939-6728-5_1, © Springer Science+Business Media LLC 2017
Chapter 1
Methods to Analyze EVs
Bernd Giebel and Clemens Helmbrecht
Abstract
Research in the field of extracellular vesicles (EVs) is challenged by the small size of the nano-sized particles.
Apart from the use of transmission and scanning electron microscopy, established technical platforms to
visualize, quantify, and characterize nano-sized EVs were lacking. Recently, methodologies to characterize
nano-sized EVs have been developed. This chapter aims to summarize physical principles of novel and
conventional technologies to be used in the EV field and to discuss advantages and limitations.
Key words Nanoparticle tracking analysis, Electron microscopy, Dynamic light scattering, Flow
cytometry, Extracellular vesicles, Resistive pulse sensing
1 Introduction
Eukaryotic and prokaryotic cells release a variety of nano- and
micron-sized membrane-containing vesicles into their extracellular
environment, which are collectively referred to as extracellular ves-
icles (EVs). EVs can be harvested from cell culture supernatants
and from all body fluids including plasma, saliva, urine, milk, and
cerebrospinal fluid [1]. Depending on their origin, different EV
subtypes can be distinguished. Together with apoptotic bodies
(1000–5000 nm), exosomes (70–160 nm) and microvesicles
(100–1000 nm) provide the most prominent groups of EVs.
Exosomes are defined as derivatives of the endosomal system and
correspond to the intraluminal vesicles of multivesicular bodies
(MVBs), which, upon fusion of the MVB with the plasma mem-
brane, are released into the extracellular environment [2–4]. In
contrast, microvesicles are directly pinched off the plasma mem-
brane [3]. Even though the release of exosomes was initially
reported in 1983 by detailed structural analysis using transmission
electron microscopy [5], research on nano-sized EVs did not gain
significant prominence until the discovery that small EVs transport
small RNAs, including micro RNAs [6, 7]. Since then, the interest](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-16-320.jpg)
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in EVs as mediators for intercellular signaling, biomarkers for
diseases, drug delivery vehicles, or therapeutical agents has dra-
matically increased [8, 9].
The research in the EV field is challenged by the small size of
the nano-sized EVs. Apart from transmission and scanning elec-
tron microscopy, established technical platforms to visualize, quan-
tify and characterize nano-sized EVs were lacking. In 2011 the
nanoparticle tracking analysis (NTA) was initially described as a
useful technology to characterize nano-sized EVs [10, 11]. NTA
has emerged as one of the most prominent, state-of-the-art tech-
nologies in the EV field. In addition, other methods adopted from
the field of nanotechnology are available, which have been or
might be used for the characterization of EVs. This chapter aims to
summarize physical principles of novel and conventional technolo-
gies to be used in the EV field and to discuss advantages and limita-
tions, which are summarized in Table 1.
Table 1
Current methods for EV analysis
Technique Particle size
Time for
measurement Limitations Advantages
Cryo-TEM 1 nm … mm 1 h Sample preparation, only
small amount of sample
is analyzed
Morphology
DLS,
homodyne
1 nm … 6 μm 1–2 min Polydisperse samples
challenging, presence of
large particles biases
results
Wide size range
DLS,
heterodyne
0.5 nm … 6 μm 1–2 min Analog homodyne DLS,
but not as dominant
Wide ranges of size and
concentration
NTA 20 nm … 1 μm 5–10 min Dilution necessary for
high concentration,
non-standardized
method
Visualization, resolution
(even polydisperse
samples), low
concentrations
FCM 300–500 nm …
10 μm
1 min Working range, pore
blocking,
calibration
Fluorescence,
biochemical
information
AFM 10–1000 nm 1 h Analog cryo-TEM Morphology
RPS 50 nm to
10 μm,
dependent on
pore size
30 min Working range, pore
blocking, calibration
High resolution,
compatible with
buffers
AF4
ca. 5 nm to
20 μm
1 h Sample dilution,
interaction of sample
with membrane
Fractionation
TEM transmission electron microscopy, DLS dynamic light scattering, NTA nanoparticle tacking analysis, FCM flow
cytometry, AFM atomic force microscopy, RPS resistive pulse sensing, AF4 flow field flow fractionation
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-17-320.jpg)
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2 EV Analysis
There are a number of optical and nonoptical methods to analyze
the size, quality, and concentration of nanoparticles. The maximal
resolution (dA) of classical light microscopy depends on the wave-
length of light (λ) and the numerical aperture (NA) of the lenses.
It can be calculated according to the formula:
dA
NA
.
=
×
l
2
(1)
High-quality lenses (e.g., oil immersion objectives) rarely reach
apertures of more than 1.4. Accordingly, at a supposed wavelength
of 550 nm, conventional light microscopes have difficulty resolv-
ing structures less than 200 nm in size. To detect smaller struc-
tures, electron microscopic techniques are required. Thus, EVs are
conventionally analyzed by electron microscopy, usually via trans-
mission electron microscopy (TEM) and in some cases by scanning
electron microscopy (SEM) [10, 12]. New fluorescence based
super-resolution microscopic techniques such as STED (stimulated
emission depletion) or PALM (photoactivated localization micros-
copy) and atomic/scanning force microscopy definitively allow for
higher resolutions and certainly will provide important informa-
tion on the nature of EVs in the near future [13–16].
For the preparation of EV samples for electron microscopy differ-
ent methods can be used. Heavy metals, such as osmium tetroxide
and uranyl acetate, increase the contrast of the analyzed samples.
However, like aldehyde-based fixation methods, heavy metal treat-
ment regularly results in the dehydration of the samples, resulting
in EV shrinkage and deformation. Accordingly, EVs frequently
adopt cup-shaped morphologies, which were initially considered as
a characteristic feature of exosomes [12]. Upon using cryoelectron
microscopic technologies lacking chemical fixation and staining
procedures, native EV sizes and shapes can almost be conserved.
Here, freshly prepared EVs are transferred to grids and immedi-
ately are cryofixed in liquid nitrogen. As a result of the procedure,
water is placed in a glass-like state without forming destructive ice
crystals, thus, leaving the EV structure largely intact [17, 18].
Although the electron microscopic analyses provide important
information on the EV morphology, this technology does not
allow EV quantification; among others EVs do not quantitatively
adhere to the grids.
Methods based on the analysis of scattered light are eminently suit-
able for the contact-free analysis of delicate samples such as bio-
nanoparticles—EVs. In nearly every analysis device, ranging from
dynamic light scattering (DLS) to fluorescent cell sorting, light
2.1 Electron
Microscopy
2.2 Physical
Background on Light
Scattering
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-18-320.jpg)
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Examples for elastic light scattering are Rayleigh scattering
and Mie scattering, which will be explained below. If the energy
of the incident and scatter light differ from each other, the pro-
cess is termed inelastic light scattering. Light scattered in a
Raman process is an example of inelastic light scattering. In
such a process a part of the energy of the incident light is trans-
formed into another form of energy, e.g., heat or vibrational
energy. In Stokes Raman scattering, the wavelength of the scat-
tered light is longer than the incident light. In anti-Stokes
Raman scattering the wavelength of the scattered light is shorter
than the wavelength of the incident light. The additional energy
derives from vibrational energy of the molecules of the particle,
e.g., when the molecules are in excited state.
Of note, compared to elastic scattering, Raman scattering
is very weak and requires well thought-out arrangements for
detection [19, 20].
The characteristic of how particles scatter light is mainly related to
their size. Within the scope of this chapter, we focus on Rayleigh
and Mie scattering.
Rayleigh scattering describes the elastic scattering of electro-
magnetic waves on particles with sizes rather small compared to
the incident wavelength r 0.2 λ. The intensity of the scattered
light is inversely related to the fourth power of the wavelength
λ of the incident light. Consequently, light with shorter wave-
lengths is scattered with higher intensities than light with lon-
ger wavelengths. A well-known phenomenon which can be
explained by Rayleigh scattering is the blue color of the sky;
2.2.3 The Influence
of Particle Size
2.2.4 Rayleigh Scattering
Fig. 1 Incident light wave with Ei and λi shifts the electrons of the particle from a
ground state E0 to a virtual level E1. Elastic scattering: from the virtual level E1,
electrons return to the ground state, no energy is transformed (e.g., Rayleigh
scattering). Inelastic scattering: electrons do not return to the ground state E0.
Parts of the incident energy (Eem″) are transformed into other energy forms (e.g.,
Stokes scattering)
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-20-320.jpg)
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molecules in the atmosphere scatter the blue parts of sunlight
approximately ten times stronger than the red parts.
The scattering intensity I also depends on the index of
refraction n of both, of the particle (n1) and surrounding medium
(n2). The refractive index is defined as the ratio between the speed
of light in a given material and in a vacuum. The relative refraction
index m=n1/n2; n1 and n2 are the refractive indices of particle and
surrounding media, respectively. Considering all these parameters,
the intensity (I) of the Rayleigh scattering at a certain distance (R)
and scattering angle (θ) [21] is given by:
I I
R
m
m
d
= ´
+ æ
è
ç
ö
ø
÷
-
+
æ
è
ç
ö
ø
÷
æ
è
ç
ö
ø
÷
0
2
2
4 2
2
2 6
1
2
2 1
2 2
cos q p
l
(3)
Of note, the intensity of Rayleigh scattering is proportional to the
sixth power of the size of small particles, which restricts the size
detection limit of many scatter based methods. In contrast, the
irradiation intensity (I0) is only linearly linked to the intensity of
Rayleigh scattering. A large difference in the refractive index of the
surrounding medium and the illuminated particles (e.g., water
n2 =1.333) increased the intensity of the scattered light.
Particles with similar or larger sizes than the wavelength of the
incident light cause Mie scattering. The formula to calculate the
intensity of Mie scattering at a given angle and distance of larger
particles is much more complex and is neglected here. Particles
with an approximate size of the wavelength of the incident light
can be considered as an aggregation of material, whose oscillating
electrons influence each other and may scatter the light toward a
certain direction. As a consequence, the Mie scattering intensity is
less dependent on the wavelength of light than Rayleigh scattering.
For example, waterdrops in clouds cause wavelength-independent
Mie scattering; that is the reason why clouds appear white.
For a more detailed description on light scattering, we like to
refer to more specific literature [22, 23].
3 Methods Based on Light Scattering
An advanced technology applying the scattering light for the
characterization of nanoparticles is the method of dynamic light
scattering (DLS), also known as photon correlation spectroscopy
(PCS). Here, a distinct proportion of the sample volume—regu-
larly a few microliters—is illuminated with a laser beam. The light
scattered from the particles within the illuminated part of the
probe is recorded over time [24]. Due to their Brownian motion,
the particles in the sample are constantly moving, some of them
leaving and some of them entering the illuminated part of the
2.2.5 Mie Scattering
3.1 Dynamic Light
Scattering (DLS)
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-21-320.jpg)
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probe. This causes fluctuations of the scattering light, which is
registered by the detector. Since smaller particles move faster
within the probe than larger particles, smaller particles cause
higher fluctuations than larger particles. By the combination of
mathematical models of the Brownian motion and the light scat-
tering theory differential particle sizes can be calculated within
seconds [25]. While in the beginning of commercial DLS (around
1970) only narrow size distributions could be measured, the
range of modern DLS instruments typically covers sizes ranging
between 1 nm and 6 μm [26]. To obtain optimal results, the
presence of contaminants such as dust particles, air bubbles,
debris and inorganic particles, which can derive from laboratory
water (e.g., silicates, phosphates, carbonates), must be circum-
vented. For better reproducibility, optimized sample preparation
including filtration of buffers is mandatory [27].
Depending on the position of the detector, two different
DLS systems are commercially available, the homodyne and the
heterodyne DLS.
In a homodyne DLS setup, the laser and detector are
arranged perpendicular to each other (Fig. 2). The incident light
with the intensity I0 illuminates the sample and becomes partially
scattered by the particles suspended in the probe. The intensity
of the scattered light (IS) is recorded by the detector. Critical
parameters in this setting are the distance the light has to pass
through the sample until it reaches the detector and the concen-
tration of the particles. If the particles are too concentrated, sec-
ondary scattering occurs diminishing the amount of scatter light
that reaches the detector. Hence, appropriate dilutions have to
be titrated to obtain valid data [28].
Within heterodyne DLS systems the backscattered light is
analyzed (Fig. 2). The incident laser light is coupled into an opti-
cal fiber to illuminate the probe with the intensity I0. Only light,
which is scattered by the particles within the probe in an angle of
180°, can reenter the optical fiber and become transmitted with
Fig. 2 Principle of homodyne and heterodyne DLS systems
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-22-320.jpg)
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an intensity IS to the detector [29]. In addition to the average size
distribution of the particles in the probe and following calibration,
heterodyne DLS regularly enables to determine the particle con-
centration of given probes. For appropriate measurements of par-
ticle sizes, analyses of polydisperse probes require particle size
differences with ratios of d1/d2 1.8 [30].
Regularly, a 20–50 μL sample volume is sufficient to determine
the average particle size distribution on commercial DLS instru-
ments in less than a minute. Analyses of monodisperse samples,
i.e., samples only containing particles with the same size, yield reli-
able results. In the case of polydisperse samples such as blood
plasma samples, the results may be less clear and require knowl-
edge of the applicable mathematical model. The results are dis-
torted by larger particles with diameters in the micrometer range,
already when they are present at low concentrations [30]. Upon
analyzing samples with high particle concentrations or samples
containing larger agglomerates, heterodyne DLS instruments pro-
vide more flexibility than homodyne instruments, but still are lim-
ited compared to other techniques such as the nanoparticle
tracking analysis (NTA) [31].
In 2011 NTA was reported to provide a suitable method for EV
characterization for the first time [10, 11]. Since then, NTA has
emerged as one of the standard techniques for the characterization
of EVs. It also allows analyses of larger particles within the microm-
eter range and thus has also been designated as particle tracking
analysis (PTA).
Analogous to DLS, NTA records the Brownian motion of
small particles. Similar to DLS, particles in the sample are visual-
ized by the illumination with incident laser light. The scattered
light of the particles is recorded with a light-sensitive CCD cam-
era, which is arranged at a 90° angle to the irradiation plane
(Fig. 3). The 90° arrangement, also known as ultramicroscopy,
allows detection and tracking of the Brownian motion of
10–1000-nm-sized vesicles. Using a special algorithm the size of
each individually tracked particle is calculated, thus simultane-
ously allowing determination of the average size distribution of
particles in a given sample as well as their concentration. Even
though the NTA technology is relatively new on the market, it
originated almost 25 years ago [32]; the commercial implementa-
tion of this technique required the availability of fast computer
systems that are able to cope with the computationally intensive
video analysis in reasonable time frames.
A brief introduction of the physical principle underlying NTA
is as follows: When small particles are dispersed in a liquid (the so-
called continuous phase, e.g., water), the particles move randomly
in all directions. This phenomenon is termed diffusion and is
expressed by the diffusion coefficient (D). In more detail, the
3.2 Nanoparticle
Tracking Analysis
(NTA)
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-23-320.jpg)
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undirected migration of given particles is caused by energy trans-
fers from surrounding water molecules to the particle. In the
absence of any concentration gradient within the dispersion and
upon long-term observation, the distances small particles move in
any direction should neutralize each other over time, leaving a
total movement of almost zero. However, during given time inter-
vals, diffusing particles move within certain volume elements. In
NTA the time t between two observation spots is quite short
(~30 ms). The distance particles have moved during the time inter-
val are recorded and quantified as the mean square displacement
(x2
). Depending on the number of dimensions (one, two or all
three dimensions) the diffusion coefficient can be calculated from
the mean square displacement as follows:
D
x
t
D
x y
t
D
x y z
t
= = =
2 2 2
2 4 6
, , ,
.
Via the Stokes-Einstein relationship, the particle diameter d can be
calculated as function of the diffusion coefficient D at a tempera-
ture T and a viscosity η of the liquid (kB Boltzmann’s constant)
[33]:
D
k T
d
=
4
3
B
.
ph
In NTA, the particle fluctuation of a single particle is registered in
two dimensions. After combining the Stokes-Einstein relationship
and the two-dimensional mean square displacement, the equation
can be solved for the particle diameter d with:
d
k T
t
t
x y
k T
x y
= × =
4
3
4 16
3
2 2
B B
.
ph ph
, ,
Fig. 3 Schematic setup of a nanoparticle tracking analyzer
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-24-320.jpg)
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By simultaneously tracking several particles, their diameters can be
determined in parallel. Figure 4 shows a typical particle size distri-
bution of vesicles harvested from blood plasma.
The lower limit of the working range, i.e., the smallest detect-
able particle size, depends on the scattered intensity of the particle
(compare Eq. 3), the efficiency of the magnifying optics and the
sensitivity of the camera [34]. Silver and gold nanoparticles are
strong scatterers due to the comparably large refractive indices of
2–4 and can be detected down to sizes of ~10 nm. Biological
nanoparticles such as EVs have refractive indices of around 1.37–
1.45 resulting in a limit of detection of 30–50 nm for NTA [35].
NTA allows the direct measurement of concentration as single
particles in the illuminated volume are visualized. Thus, NTA is an
absolute measurement technique allowing the determination of
total surface or volumes of particles in a sample (see Fig. 4). For the
measurement of concentration, the instrument is calibrated with
Fig. 4 Particle size distributions of vesicles in blood plasma. The particle size distributions range from 100 to
1000 nm dependent on weighing according to number, area, or volume. NTA as absolute technique allows
quantification of concentration, area, and volume of vesicles present in the sample
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-25-320.jpg)
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size standards of known size and concentration. The visualization
of the sample gives a unique impression on the quality of the sam-
ple, such as the presence of agglomerates. The working range of
0.5×106
and 1×1010
particles per cm3
is very low compared to
DLS, allowing NTA to analyze low concentrated samples. To
record representative size distribution profiles, it is recommended
to analyze a range of 1000–10,000 single particles.
While in the early stages of NTA development, the manual
adjustment of microscope and laser was time-consuming, nowa-
days, the measurement cell is aligned within minutes. Currently,
commercial NTA instruments are offered by only two companies
(Malvern Instruments Ltd. and Particle Metrix GmbH).
Depending on the model temperature control, conductivity and
zeta potential measurement are integrated. The zeta potential
reflects the surface charge of given particles, which might be
related to their stability. Currently, efforts are undertaken to
implement additional components, which, for example, can auto-
matically dilute probes to optimal particle concentrations, record
electrochemical parameters (e.g., the pH of the probe), and allow
for the specific characterization of fluorescent-labeled EVs.
The quality of an NTA result is influenced by particle con-
tamination. In addition to the contaminating particles, which
were mentioned in the section of DLS, high concentrations of
stabilizing agents (e.g., surfactants) are critical as soon as they
reach their critical micellar concentration (CMC). Contaminating
particles may derive from diluents (distilled water or buffer agents)
or from chemicals used during preparation of samples. Regularly,
chemicals are not certified for the absence of nanoparticles.
Precipitates of phosphates, carbonates, or silicates as well as dust
can be removed by filtration of the buffers, ideally with pore sizes
below 50 nm. Degassing in ultrasonic bath is also helpful to
remove air bubbles [34].
For the characterization of EVs, it would be desirable to simul-
taneously analyze the presence of different molecules expressed
on the surface of EVs using a high-throughput technology. At
the cellular level, such analyses are regularly performed by
FC. However, due to the configuration of conventional flow
cytometers, the size detection limits of particles lie somewhere
between 300 and 500 nm [36]. Thus, by means of conventional
flow cytometry, only large EVs can be analyzed at an individual
particle level. To this end, EV FC analyses have indeed already
been carried out on larger EVs, particularly in the area of plate-
let research. In the literature corresponding EVs are usually
referred to as microparticles [37–39]. Analyses of smaller EVs
by flow cytometry require either a special mechanical setup, or
EVs must be bound by immunological methods to carrier
particles.
3.3 Flow
Cytometry (FC)
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-26-320.jpg)
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Magnetic carrier particles or latex beads can be coated with
antibodies that recognize epitopes on EVs, e.g., anti-CD63 anti-
bodies. If the antibody-coated beads are added to EV-containing
samples, aggregates between the beads and the EVs are formed,
which can be concentrated by magnetic separation or by low-speed
centrifugation, respectively. For an appropriate aggregation, suffi-
cient quantities of EVs need to be present in the sample; the beads
should get saturated with EVs, otherwise aggregates with several
beads might form. The aggregate formation of EVs with several
beads can be reduced by vortexing or pipetting. In analogy to cells,
the formed bead-EV aggregates can be labeled with different
fluorescence-
labeled antibodies. Due to the presence of the beads,
these aggregates are big enough to be analyzed on conventional
flow cytometers [40–43]. This technology offers the great advan-
tage for a fast and comprehensive EV characterization. However,
since only aggregates and not individual EVs are analyzed, this
form of analysis is a bulk analysis and finally may not reveal much
more information than conventional Western blots.
Irrespective of the low size resolution of conventional flow
cytometers, analyses of small EVs at the single-particle level pro-
vide several challenges. As long as the particles are larger than the
wavelength of light, their size corresponds to the amount of the
forward-scattered light, which is measured at the forward scatter
detector. If the particle sizes are around or below the wavelength
of the light, the intensity of light scattered to the side increases
proportionally to the forward-scattered light. Accordingly, the size
of particles that are smaller than the wavelength of the incident
light can better be determined upon measuring the scattered light
at the side scatter detector than on the forward scatter detector.
Alternatively, an extended forward scatter detector can be used,
which collects the forward-scattered light and proportions of the
side scattered light.
Groups that have optimized the setup of configurable flow
cytometers for the measurement of nano-sized particles were
already able to analyze viruses and EVs at a single-particle resolution
[44–46]. Essential prerequisites for such measurements are the
reduction of signal-to-noise ratio and an increase in the sensitivity
of the scatter light detection. According to the formula of the
Rayleigh scattering, a linear increase in sensitivity can be achieved
by increasing the intensity of the laser light [44]. In addition, the
signal-to-noise ratio largely depends on the processing of the
sheath fluid. Regularly, commercial products are sterilized by filtra-
tion through 0.22 μm filters, which is not sufficient to remove
background noise producing nanoparticles such as calcium phos-
phate or calcium carbonate nanoparticles. Thus, filtration through
0.05 μm filters is highly recommendable [44]. The background
noise can also be reduced upon staining EVs with a strong fluores-
cent dye, e.g., the membrane-intercalating PKH67, and by trig-
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-27-320.jpg)
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gering the subsequent flow cytometric measurements on the
fluorescence and not as conventionally on the scattered light [46].
The disadvantage here is that aggregates of the unbound fluoro-
chromes should be removed before stained EVs get analyzed. Even
though it is time consuming, currently, density gradient centrifu-
gation appears as the most appropriate technology to separate fluo-
rochrome aggregates and stained EVs. Irrespectively of this, EVs
can also be marked with fluorescence conjugated antibodies allow-
ing for the specific analyses of antigens of interest [46, 47]. Since
the surface of EVs is orders of magnitude smaller than that of cells,
antibodies should be used being conjugated to very bright fluoro-
chromes such as B-phycoerythrin (B- PE) or R-PE. Usage of anti-
bodies with weaker fluorochromes can only be recommended,
when corresponding epitopes are known to be expressed on the
EVs very abundantly [47].
Another challenge is the concentration of the EVs to be mea-
sured. Ideally, for single particle analyses, the concentration of par-
ticles to be measured should be in the range of 5×105
to 5×106
particles per ml sample liquid. If particles are higher concentrated,
swarm detection can occur, that is, the simultaneous detection of
several particles at a given moment [48]. Following enrichment of
EVs, the concentration regularly strongly exceeds this value; con-
sequently, probes to be measured have to be diluted to sometimes
homeopathic appearing dilutions.
Raman scattering is a form of inelastic light scattering [19]. Even
though most of the incident light is scattered in an elastic manner,
each molecule also specifically scatters light in an inelastic manner and
thus generates individual Raman spectra of the scattered light. Raman
microspectroscopy allows the recording and analysis of sample spec-
tra and thus gives information on molecular composition of probes of
interest. This technique has been used to analyze the composition of
EVs and allowed discrimination of different EV subtypes from each
other [49]. Especially when combined with atomic force microscopy,
Raman spectroscopy might offer a very potent technology to analyze
and discriminate different EV subtypes [50].
Raman microspectroscopy is a relatively high-priced and spe-
cialized technique. Setup and acquisition require a relatively large
amount of time, resulting in an incompatibility with high-
throughput analyses (10–100 vesicles per hour). Due to the low
intensity of the Raman scattering signal (approx. 1:10,000 of
elastic scattering), the measurement is influenced by artifacts
demanding high grade of manual effort and expertise of the oper-
ating personnel. During measurement, EVs are exposed to a high-
intensity light beam, which can induce photostress and cause
adverse effects. Depending on the dose and wavelength of the
incident light beam, (photo) reactions might be induced in the
EVs and change them irreversibly [49].
3.4 Raman
Microspectroscopy
(RM)
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-28-320.jpg)
![14
In the 1980s, considerable efforts were made to develop tech-
niques allowing resolving solid state surfaces at atomic levels. As a
result, the atomic force microscope (AFM) [51] and later the scan-
ning tunneling microscope (STM) were developed.
AFM is based on a tip mounted on a cantilever that is moved like
the pick-up of a record player in a defined distance over the surface of
the material to be analyzed. The radius of the tip ideally is reduced to
that of a few atoms. The torsion of the cantilever is a measure for the
forces between tip and surface as function of the distance. The tip is
either attracted (e.g., van der Waals forces) or repelled (e.g., electro-
static forces) from the surface resulting in characteristic force-distance
curves. In the beginning, AFM has been utilized for the quantitative
description of the topology of solid-state surfaces under vacuum con-
ditions. Meanwhile immobilized particles such as vesicles can also be
analyzed in buffers [36, 52, 53]. Thus, AFM became a feasible
method for the characterization of EVs, especially to analyze their
size and topology [54, 55]. However, as immobilization of EVs
might affect their topology, results are influenced by the mode of
sample preparation [56].
Resistive pulse sensing (RPS) is a technology to measure absolute
sizes and the concentration of particles in suspension, whose sizes
range from 100 nm to 100 μm. In principal, the system contains
two cells, both equipped with an electrode. The cells are con-
nected by membrane containing a small pore or a micro-channel,
regularly with pore sizes below 1 μm (Fig. 5). To analyze the
particle concentration and the average size distributions of sus-
pensions, an electric field is applied onto the electrodes. As a con-
sequence, charged particles migrate to the anode or cathode,
respectively. In analogy to the Coulter principle, each time a par-
ticle passes through the pore, the electrical resistance of the buf-
fer gets altered. These alterations in resistance are recorded. Since
alterations in the resistance depend on the volume of the migrat-
ing particles, the particle sizes and their zeta potential can be
calculated [57]. As a prerequisite for this method, the pore diam-
eter (q) has to be much smaller than the pore thickness (l).
Following calibration with particles of defined sizes, particle sizes
and their zeta potentials can be calculated; they are proportional
to the shapes and heights of the recorded pulses. Considering the
pore of the membrane as a cylinder, the electrical resistance (R)
of the pure buffer can be calculated as:
R
l
A
= r
ρ: specific resistance of the buffer, l: pore thickness (typically
several tens of μm), A: pore area.
3.5 Scattered-Light-
Independent
Technologies
3.5.1 Atomic Force
Microscopy (AFM)
3.5.2 Resistive Pulse
Sensing (RPS)
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-29-320.jpg)
![15
With A=π/4×q2
the pore area is related to the pore diameter
(q). In reality, each particle contains a specific electrical resistance
which theoretically has to be considered. However, specific electrical
resistances of given particles are high. If particles are considered as
insulators, their specific electrical resistance can be neglected [58].
Provided the platform is equilibrated with particles of known
concentration, the estimated count rates of given particle suspen-
sions to be analyzed reveal their particle concentrations.
The upper end of the working range is limited by the pore size,
the lower end on the sensitivity in the detection of resistance changes
(typically ~0.2 q). Before usage, every membrane needs to be cali-
brated with size standards. Upon analyzing biological samples, pore
blocking often increases the analysis time per sample of up to 1 h.
RPS instruments capable of detecting particles in the lower nanome-
ter ranges (in general 100 nm) are under development [59].
The family of field flow fractionation techniques (FFF) comprises
instruments separating polydisperse samples in individual fractions
while simultaneously determining their particle size. FFF techniques
are characterized by high resolution and compatibility to flow detec-
tors and have already been used to characterize EVs [60, 61].
The separation is based on the so-called cross-flow principle,
in which two orthogonal forces act on the particle. Depending on
the underlying FFF technique, the forces can be created differ-
ently, either by friction in flow field FFF (FFFF, F4
), sedimentation
(sedimentation FFF, SdFFF), or an electrical field (ElFFF). FFFF
and SdFFF are the most common techniques [62].
In FFFF a separation channel with an asymmetrical flow pro-
file (asymmetrical FFFF, AF4
) is prevailingly used; it gives the
most reproducible results with lowest sample loss (Fig. 6). Before
3.5.3 Field Flow
Fractionation (FFF)
Fig. 5 Resistive pulse sensing (RPS). Left: Typical setup with two cells separated
via an insulating membrane with a single pore. Right: Transient signal of current
representing a (1) large-, (2) small-, and (3) medium-sized particle. Following
calibration, the count rate, i.e., the number of pulses per time interval, reflects
the concentration of the particle suspension to be analyzed
Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-30-320.jpg)
![16
fractionation starts, the sample is focused as a small band on the
semipermeable membrane. During fractionation two perpendicu-
lar flows act on the sample, a laminar flow and a cross flow. The
cross flow counteracts the diffusion tendency of the particles
away from the membrane. Approximately 10 min after starting
the measurement, the diffusion and cross flow are equilibrated
and the particles have accumulated in a certain distance to the
membrane, which depends on the diffusion coefficient of the par-
ticles. Since smaller particles contain higher diffusion coefficients
than larger ones, smaller particles accumulate at higher distances
away from the membrane than larger particles. The laminar flow
transports particles to the detector. The closer particles accumu-
late toward the middle of the flow channel, the faster they are
transported to the detector. As a result, smaller particles arrive
earlier at the detector than larger ones. For the detection a single
detector or a combination of detectors can be used. The follow-
ing detectors are available: absorption detector (diode array),
fluorescence detector, scattering light detector, and atom spec-
troscopic detector (e.g., inductively coupled plasma mass spec-
trometer, ICP-MS). The usage of detectors allowing deciphering
the chemical composition of probes (HPLC, Raman, TXRF) has
been reported [63].
Typically, polydisperse samples containing three or more dif-
ferent components can be separated. The injection volumes depend
on the type of sample and the concentration of its particles; it may
vary from between 20 μl to 2 ml. To set up a successful separation
composition, concentration and pH of eluents need to be optimized
to prevent aggregation or irreversible binding of the particles to
the membrane [64]. During separation the particles are regularly
diluted 100- to 1000-fold.
Fig. 6 Asymmetrical flow field flow fractionation (AF4
). Before separation, given samples are concentrated on
the membrane. Depending on their sizes, particles diffuse from the membrane and accumulate at certain posi-
tions in which equilibriums of diffusing and cross flow forces are given. Simultaneously to their diffusion, the
laminar flow transports the particles toward the detector at the end of the flow channel
Bernd Giebel and Clemens Helmbrecht](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-31-320.jpg)
![17
4 Conclusion
EVs can be considered as extracellular signal organelles which
mediate intercellular communications. Accordingly, they are essen-
tially involved in normal physiological and pathophysiological pro-
cesses. In addition to its basic scientific importance, the young field
of EV research offers an extremely high innovation potential for
novel diagnostic and therapeutic procedures. Although the num-
ber of EV publications has tremendously increased in recent years,
progress in the field of EV research is limited by the lack of stan-
dardized methods for their analyses as well as for their processing.
Interdisciplinary collaborations of device developers and compa-
nies with scientists will certainly help to overcome these limitations
within the next few years and surely will give new impacts on the
exciting field of EV research.
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![19
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Methods to Analyze EVs](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-34-320.jpg)
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Chapter 2
Tunable Resistive Pulse Sensing for the Characterization
of Extracellular Vesicles
Sybren L.N. Maas, Marike L.D. Broekman, and Jeroen de Vrij
Abstract
Accurate characterization of extracellular vesicles (EVs), including exosomes and microvesicles, is essential
to obtain further knowledge on the biological relevance of EVs. Tunable resistive pulse sensing (tRPS) has
shown promise as a method for single particle-based quantification and size profiling of EVs. Here, we
describe the technical background of tRPS and its applications for EV characterization. Besides the stan-
dard protocol, we describe an alternative protocol, in which samples are spiked with polystyrene beads of
known size and concentration. This alternative protocol can be used to overcome some of the challenges
of direct EV characterization in biological fluids.
Key words Extracellular vesicles, Exosomes, Microvesicles, Characterization, Quantification, Size
distribution, qNano, Resistive pulse sensing
1 Introduction
Due to their small size (50–1000 nm), accurate characterization of
extracellular vesicles (EVs) is technically challenging. Over time,
different techniques have been developed to overcome these chal-
lenges. Most of these techniques are based on bulk analysis of EVs.
For instance by total protein quantification, western blotting,
bead-based flow cytometry [1] or modified protein microarrays
[2]. However, alternative techniques, that allow for single particle
analysis of EVs, have become recently available [3–8]. One of those
techniques, provided by the qNano platform (Izon Science Ltd), is
tunable resistive pulse sensing (tRPS) (Fig. 1).
In tRPS, a non-conductive membrane (“nanopore”) separates
two fluid cells [9] (Fig. 2). This nanopore is punctured to create a
single conical shaped opening (Fig. 2, top-left). Once a voltage is
applied, a current of charged ions through the nanopore is estab-
lished. This baseline current is distorted, as observed by the appear-
ance of peaks or “pulses,” as particles move through the nanopore
(Fig. 2, bottom). Once a particle enters the sensing zone of the
Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545,
DOI 10.1007/978-1-4939-6728-5_2, © Springer Science+Business Media LLC 2017](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-36-320.jpg)
![23
nanopore [10] (Fig. 2, timing 1), the flow of charged ions, and thus
the baseline current, will be altered (Fig. 2, top-right). As the par-
ticle enters the conical opening, the relative blockade of the baseline
current will be maximum (Fig. 2, timing 2). This blockade will
gradually decrease to baseline levels as the particle moves further
through the nanopore (Fig. 2, timing 3). To characterize particles
in a sample, a calibration sample of (polystyrene) beads of known
volume and concentration is measured first. The magnitude of
pulses and the particle rate induced by this reference sample can
subsequently be used to calculate the size profile and concentration
of the particles in the measurement sample [11, 12].
The movement of particles through the nanopore is based on
several independent forces, being electrokinetic (electrophoretic
and electro-osmotic) and fluidic forces [10]. The variable pres-
sure module (VPM) can be used to apply additional external
force and should be used (≥0.8 kPa) to minimize interfering
electrokinetic forces when analyzing particles using the smaller
(NP100-NP200) nanopores [13].
Characterization of EVs using tRPS is technically challenging.
Due to the heterogeneous nature of EVs a large size range of parti-
cles is usually present in a sample. Larger-sized EVs may clog the
nanopore, thereby obstructing the measurement. Secondly, the
sample with calibration beads should consist of the same buffer com-
ponents as the EV sample. This may be technically unfeasible, as the
buffer components are regularly unknown when measuring EVs,
especially when measuring EVs directly in a biological sample. This
problem can be overcome by using a “spiking” approach, in which
the calibration beads are added to the measurement sample [3].
Here, we describe two different approaches for the characteriza-
tion of EVs using tRPS. First, the standard protocol is described,
which often suffices for the characterization of purified EVs.
Secondly, we describe the alternative spiking approach, which could
be of benefit when characterizing EVs in biological samples.
2 Materials
1. qNano instrument (Izon Science Ltd, Christchurch, New
Zealand).
2. Variable Pressure Module (Izon Science Ltd, Christchurch,
New Zealand).
3. Polystyrene calibration particles (Izon Science Ltd,
Christchurch, New Zealand) (see Note 1).
4. Nanopores (Izon Science Ltd, Christchurch, New Zealand)
(see Note 2).
2.1 qNano Specific
Equipment/Materials
tRPS for EV Characterization](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-38-320.jpg)
![25
8. Apply ≥0.8 kPa pressure using the VPM and click “record”
(see Note 13). Make sure that a particle rate (see Note 14) of
100 min−
and a mode blockade height of 0.1 nA is recorded
(see Note 12).
9. If the baseline current suddenly drops or keeps drifting during
recording, pause the recording and try to reestablish a stable
current (see Note 9).
10. Record 500 particles, for at least 30 s (see Note 14). Fill out
the details of the calibration sample in the pop-up form.
11. Optionally, multi-pressure measurement can be performed (see
Notes 13 and 15). Hereto, add at least 0.2 kPa and record a
second measurement (more steps could increase accuracy).
12. Remove the calibration sample and wash the upper fluid cell by
resuspending 100 μl PBS in the upper fluid cell 3–4 times.
Remove residual PBS by usage of the lint-free tissue (see Note
16).
13. Introduce the EV sample and make sure the baseline current is
within 3% of the baseline for the calibration sample (see Note
17).
14. Record the sample at the same VPM pressure(s) as applied for
the calibration sample.
15. Click the “Analyse data” tab and right-click on “Unprocessed
files” and select “Process files”.
16. Click on the checkbox in the “calibrated” column next to one
of the sample files. This will initialize the calibration pop-up
menu. Select the “multi-pressure measurement” tab if appli-
cable and select the sample files and calibration file(s).
17. Once calibrated, an EV sample file will display a size distribu-
tion in nm instead of nA (Fig. 3, right). Click on “Preview” to
generate a .pdf file containing statistics such as the concentra-
tion (measured and raw if a diluted sample was used).
The standard protocol for tRPS-based EV quantification relies on
usage of appropriately formulated calibration samples (i.e., with the
diluents resembling the fluid of the EV sample). This may be unfea-
sible for biological fluids, since their exact composition may be
unknown rendering their simulation impossible. Secondly, the vol-
ume of the biological sample (e.g., only 100 μl of plasma) may be
insufficient for preparation of calibration fluid (which usually can be
done by removal of small particulate matter by ultracentrifugation
or filtering). In such cases, an alternative is provided by performing
a spiking protocol, in which calibration beads are introduced in the
EV sample [3]. This methodology can also be used when samples
are measured over a prolonged period of time and stable nanopore
conditions cannot be guaranteed due to nanopore clogging.
3.2 Spiking
the Sample
with Polystyrene
Beads of Known Size
and Concentration
tRPS for EV Characterization](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-40-320.jpg)
![30
(RMS) noise should be 10 pA. If these conditions are not met,
air bubbles or (partial) nanopore blocking may be causative. To
solve this, resuspend the sample in the upper fluid cell and check
if the baseline becomes stable. If not, remove both the sample
and the PBS in the lower fluid cell. If (after reapplication of
PBS) no stable baseline current is established, the nanopore
may be (partially) blocked. Tap the shielding cap (using the sup-
plied plunger) to vibrate the nanopore and to disrupt particles.
Clogging may also be solved by induction of a brief pressure by
pushing down and pulling out of the plunger. Alternatively, the
shielding cap can be put in place whilst pressing on the nano-
pore, which will vibrate the nanopore. Also, the nanopore can
be maximally stretched (i.e., 47 mm), combined with applying
maximal external pressure. If still unsuccessful, remove the
nanopore and rinse heavily using deionized water. Re-place the
nanopore on the instrument.
10. Each nanopore has a target concentration. For the NP100 and
NP150 nanopores the target concentration is 10E10 per ml
and for the NP200 the target concentration is 10E9 per ml.
11. The blockade height caused by a particle moving through the
nanopore is based on the stretch, the applied voltage and the
buffer used. If the nanopore opening is reduced (less stretch
applied) the relative blockade by the particle will increase. This
also implies that smaller particles surpass the detection thresh-
old. Larger particles, on the other hand, will block the nano-
pore more frequently. By increasing the voltage applied, the
flow of ions will increase and so will the (relative) blockade
caused by particles moving through the nanopore. However,
an increased voltage can also result in increased RMS noise.
The flow of ions, and thus a higher baseline current, can also
be established by using a buffer with increased salt concentra-
tion. However, this may influence the EV characteristics, for
instance caused by changes in osmosis.
12. For accurate detection of particles a mode blockade of at least
0.1 nA is required. However, the mode blockade set for the cali-
bration particles will also determine the range of EVs detectable
by the instrument. For instance, a mode blockade of 0.1 nA for
203 nm calibration beads indicates that the instrument will only
be able to detect particles that are slightly smaller than 203 nm.
Reducing the stretch or increasing the voltage (see Note 11)
could be needed to decrease the lower detection limit.
13. External pressure needs to be applied to counteract the influ-
ence of electrokinetic forces. These electrokinetic forces are
not negligible when using small pore sizes (NP100-NP200)
[14], which is often the case upon EV quantification. Since
EVs display a modest zeta potential (i.e., the potential
Sybren L.N. Maas et al.](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-45-320.jpg)
![31
difference between the dispersion medium and the stationary
layer of fluid attached to the particle) [15, 16] the influence of
the electrokinetic forces is low and can be completely abol-
ished by applying 0.8 kPa external pressure [13].
14. The particle rate recorded (particles per minute) will depend on
the concentration of the particles, the applied pressure and
applied stretch (the rate will decrease by decreasing the stretch).
Since at least 500 particles should be recorded, a particle rate of
100 per minute is advised but not required. In our experience
particle rates 2000 per minute will be less reliable.
15. Multi-pressure measurement is advisable when measuring EVs
with increased surface charge (e.g., as a result of coupling highly
charged ligands to the surface). In such cases, difference in sur-
face charge between EVs and polystyrene calibration beads will
result in inaccurate concentration estimations as one of the par-
ticle sets is more likely to move through the nanopore than the
other [14]. Measurement of the calibration bead sample and
EV sample at multiple pressures provides additional data that is
used to accurately calculate the concentration of EVs.
16. Residual PBS in the upper fluid cell can dilute the measurement
sample. To prevent this, remove the upper fluid cell and gently
wipe lint-free tissue in the bottom-opening of the cell.
17. To accurately compare a calibration sample with an EV sam-
ple, the baseline current should not differ more than 3%. If
unable to reach a comparable baseline current, apply the
strategy outlined in Note 9. Alternatively, dilution of the sam-
ple in PBS could make the EV sample more comparable to
the calibration sample.
18. Dilution in PBS may facilitate EV counting by the qNano
instrument. However, to guarantee appropriate counting of
EVs, try to keep the particle rate above 70–100 particles per
minute (see Note 14).
19. Although not strictly necessary, an EV-to-bead ratio of approx-
imately 1 will make the measurements most reliable. If EVs or
beads outnumber their counterparts the calculation of con-
centrations will be more prone to variation.
20. To distinguish EVs from polystyrene beads, both populations
should be identifiable based on blockade sizes. For EV quantifi-
cations in biological samples we tend to use a NP200
nanopore
in combination with CPC400 (mode 335 nm) polystyrene
beads or an NP150 nanopore in combination with 203 nm
beads. To maximize the population of EVs detected, try to
obtain settings where the polystyrene beads induce blockades of
at least 0.5 nA. By increasing the blockade height caused by the
polystyrene particles, the detection limit for the EVs will
decrease (see Note 12).
tRPS for EV Characterization](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-46-320.jpg)
![35
Chapter 3
Immuno-characterization of Exosomes Using Nanoparticle
Tracking Analysis
Kym McNicholas and Michael Z. Michael
Abstract
Due to their size, small extracellular vesicles such as exosomes have been difficult to identify and to quan-
tify. As the roles that exosomes play in intercellular signalling become clearer, so does their potential utility
as both diagnostic biomarkers for disease and as therapeutic vectors. Accurate assessment of exosomes,
both their number and their cargo, is important for continued advancement in the field of vesicle research.
To that end, several technologies, including nanoparticle tracking analysis, have been developed to define
the physical characteristics of vesicle preparations and determine their concentration. This chapter describes
a method for identifying the size and concentration of a subpopulation of vesicles in biological samples,
using nanoparticle tracking analysis. Characterization of distinct exosomes is enabled by specific marker
antibodies, coupled to fluorescent quantum dots.
Key words Quantum dots, Qdots®
, NanoSight, CD63, Exosomes, Microvesicles
1 Introduction
Exosomes are extracellular vesicles, released from a wide variety of
cell types, and are being increasingly recognized as mediators of
intercellular signaling and potential biomarkers of human disease
[1]. They comprise a specific subset of cell-derived vesicles, formed
through the endosomal pathway [2]. The precise definition of an
exosome is still open to some debate, even regarding their size.
Early exosomal research was based on electron micrographs show-
ing cup-shaped vesicles up to 100 nm in diameter, but more recent
work has widened this range from 50-180 nm [3–5]. Regardless, it
is their small size that poses unique challenges for their study.
Nanoparticle tracking analysis (NTA) has been recently devised as a
means of both quantifying and determining the size of particles
between 30 nm and 1 μm in diameter. This analysis is reliant on a
NanoSight (NanoSight Limited, Malvern Instruments, Amesbury,
Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545,
DOI 10.1007/978-1-4939-6728-5_3, © Springer Science+Business Media LLC 2017](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-49-320.jpg)
![[1] A hill near Paris famous for asses.
Another time, while we were in the field, commanded by the great
Sultan Erguebzed in person, and I, harassed by a forced march, was
taking a nap in my tent, I thought I had the conclusion of an
important affair to sollicite in the divan: I went to appear before the
council of regency: but you may judge how much I had reason to be
surprized. I found the hall full of racks, troughs, mangers, and coops
for fowls; in the great Seneschal's easy chair I saw but an ox
chewing the cud; in the Seraskier's place, a Barbary sheep; on the
Testesdar's bench, an eagle with a hooked bill and long talons;
instead of the Kiaja and Kadilesker, two large owls cloathed in fur;
and for Visirs, geese with peacocks tails. I presented my petition,
and instantly heard a horrible racket, which awaked me.
Is that a dream of very difficult interpretation? said Mangogul,
you had at that time some affair in the divan, and before you went
thither, you took a walk to the Menagerie: but Signor Bloculocus, you
tell me nothing concerning my dog's head.
Prince, answer'd Bloculocus, 'tis a hundred to one, that madam
wore, or you had observed some other lady wear a sable tippet; and
that the first Dutch dog, which you saw, struck your imagination.
There you have ten times more connections than is requisite to
employ your mind during your sleep: the resemblance of colour
made you substitute hair for a tippet, and in an instant you planted
an ugly dog's head in the place of a very beautiful woman's head.
Your notions to me appear just, replied Mangogul: why do you
not publish them? they may contribute to the progress of divination
by dreams, an important science, which was much cultivated two
thousand years ago; and has since been too much neglected.
Another advantage of your system is, that it would not fail throwing
light on several works, both ancient and modern, which are but a
string of dreams; such as Plato's treatise of idea's, the fragments of
Hermes Trismegistus, the literary paradoxes of father Harduin, the
Newton, the optic of colours, and the universal mathematicks of a
certain Bramin. For example, would you not inform us, Mr. Conjurer,](https://image.slidesharecdn.com/53738-250404111014-4a18c8bb/85/Exosomes-and-Microvesicles-Methods-and-Protocols-1st-Edition-Andrew-F-Hill-Eds-51-320.jpg)
Exosomes and Microvesicles: Methods and Protocols 1st Edition Andrew F Hill (Eds.)
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- 5. Exosomesand Microvesicles Andrew F. Hill Editor Methods and Protocols Methods in Molecular Biology 1545
- 6. Me t h o d s i n Mo l e c u l a r Bi o l o g y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 7. Exosomes and Microvesicles Methods and Protocols Edited by Andrew F. Hill DepartmentofBiochemistryandGenetics,LaTrobeInstituteforMolecularScience, LaTrobeUniversity,Bundoora,VIC,Australia
- 8. ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6726-1 ISBN 978-1-4939-6728-5 (eBook) DOI 10.1007/978-1-4939-6728-5 Library of Congress Control Number: 9781493967261 © Springer Science+Business Media LLC 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Editor Andrew F. Hill Department of Biochemistry and Genetics La Trobe Institute for Molecular Science La Trobe University Bundoora, VIC, Australia
- 9. v Exosomes and Microvesicles: Methods and Protocols brings together a collection of methods for studying extracellular vesicles (EV). There has been significant growth in the field of EV research over the last decade as we understand more about the role of exosomes, microves- icles, and other EVs in many facets of cellular biology. This has been brought about with the emerging role of EVs in cell-cell communication and their potential as sources of dis- ease biomarkers and a delivery agent for therapeutics. The protocols in this volume of Methods in Molecular Biology cover methods for the analysis of EVs which can be applied to those isolated from a wide variety of sources. This includes the use of electron microscopy, tunable resistance pulse sensing, and nanoparticle tracking analysis. Furthermore, analysis of EV cargoes containing proteins and genomic material is covered in detailed chapters that contain methods for proteomic and genomic analysis using a number of different approaches. Also presented are approaches for isolating EVs from different sources such as platelets and neuronal cells and tissues. Combined these provide a comprehensive discussion of relevant methodologies for researching EVs. As with other volumes in the Methods in Molecular Biology series, the notes sections at the end of each methods chapter give invaluable insight into the methods and provide information which can help with troubleshooting and further experimental optimization. I would like to thank the chapter authors for their contributions to this volume and the editorial assistance of John Walker (Series Editor) in putting this volume together. Melbourne, Australia Andrew F. Hill Preface
- 10. vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Methods to Analyze EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Bernd Giebel and Clemens Helmbrecht 2 Tunable Resistive Pulse Sensing for the Characterization of Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Sybren L.N. Maas, Marike L.D. Broekman, and Jeroen de Vrij 3 Immuno-Characterization of Exosomes Using Nanoparticle Tracking Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Kym McNicholas and Michael Z. Michael 4 Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Romain Linares, Sisareuth Tan, Céline Gounou, and Alain R. Brisson 5 Quantitative Analysis of Exosomal miRNA via qPCR and Digital PCR . . . . . . . 55 Shayne A. Bellingham, Mitch Shambrook, and Andrew F. Hill 6 Small RNA Library Construction for Exosomal RNA from Biological Samples for the Ion Torrent PGM™ and Ion S5TM System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Lesley Cheng and Andrew F. Hill 7 A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration . . . . . . . 91 Rong Xu, Richard J. Simpson, and David W. Greening 8 Multiplexed Phenotyping of Small Extracellular Vesicles Using Protein Microarray (EV Array) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Rikke Bæk and Malene Møller Jørgensen 9 Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Karine Laulagnier, Charlotte Javalet, Fiona J. Hemming, and Rémy Sadoul 10 A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Rocío Perez-Gonzalez, Sebastien A. Gauthier, Asok Kumar, Mitsuo Saito, Mariko Saito, and Efrat Levy 11 Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Pascal Leblanc, Zaira E. Arellano-Anaya, Emilien Bernard, Laure Gallay, Monique Provansal, Sylvain Lehmann, Laurent Schaeffer, Graça Raposo, and Didier Vilette
- 11. viii 12 Isolation of Platelet-Derived Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . 177 Maria Aatonen, Sami Valkonen, Anita Böing, Yuana Yuana, Rienk Nieuwland, and Pia Siljander 13 Bioinformatics Tools for Extracellular Vesicles Research . . . . . . . . . . . . . . . . . . 189 Shivakumar Keerthikumar, Lahiru Gangoda, Yong Song Gho, and Suresh Mathivanan 14 Preparation and Isolation of siRNA-Loaded Extracellular Vesicles . . . . . . . . . . . 197 Pieter Vader, Imre Mäger, Yi Lee, Joel Z. Nordin, Samir E.L. Andaloussi, and Matthew J.A. Wood 15 Interaction of Extracellular Vesicles with Endothelial Cells Under Physiological Flow Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Susan M. van Dommelen, Margaret Fish, Arjan D. Barendrecht, Raymond M. Schiffelers, Omolola Eniola-Adefeso, and Pieter Vader 16 Flow Cytometric Analysis of Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . 215 Aizea Morales-Kastresana and Jennifer C. Jones Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Contents
- 12. ix Maria Aatonen • Division of Biochemistry and Biotechnology, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Samir E.L. Andaloussi • Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden Zaira E. Arellano-Anaya • IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France Rikke Bæk • Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark Arjan D. Barendrecht • Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands Shayne A. Bellingham • Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC, Australia; Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia Emilien Bernard • Hôpital Neurologique Pierre Wertheimer, Bron-Lyon, France Anita Böing • Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Alain R. Brisson • Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Pessac, France Marike L.D. Broekman • Department of Neurosurgery, University Medical Center Utrecht, Utrecht, The Netherlands; Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands Lesley Cheng • Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC, Australia; Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia S.M. van Dommelen • Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands O. Eniola-Adefeso • Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA M. Fish • Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA Laure Gallay • CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, France; Institut NeuroMyoGène (INMG), CNRS UMR5310 – INSERM U1217, Université de Lyon – Université Claude Bernard, Lyon, France Lahiru Gangoda • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia S.A. Gauthier • Department of Psychiatry, New York University Langone Medical Center, Orangeburg, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, Orangeburg, NY, USA; Division of Analytical Psychopharmacology, Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA; Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA Contributors
- 13. x Yong Song Gho • Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea Bernd Giebel • Institute for Transfusion Medicine, University Hospital Essen, University Duisburg-Essen, Essen, Germany Céline Gounou • Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Pessac, France David W. Greening • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia Clemens Helmbrecht • Particle Metrix GmbH, Meerbusch, Germany Fiona Hemming • Equipe 2, Neurodégénérescence et Plasticité, INSERM, U836, Grenoble, France; Grenoble Institute of Neuroscience, Université Joseph Fourier, Grenoble, France Andrew F. Hill • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia Charlotte Javalet • Equipe 2, Neurodégénérescence et Plasticité, INSERM, U836, Grenoble, France; Grenoble Institute of Neuroscience, Université Joseph Fourier, Grenoble, France Jennifer C. Jones • National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; Molecular Immunogenetics and Vaccine Research Section Vaccine Branch, CCR, Bethesda, MD, USA Malene Møller Jørgensen • Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark Shivakumar Keerthikumar • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia A. Kumar • Department of Psychiatry, New York University Langone Medical Center, Orangeburg, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, Orangeburg, NY, USA; Division of Analytical Psychopharmacology, Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA; Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA Karine Laulagnier • Equipe 2, Neurodégénérescence et Plasticité, INSERM, U836, Grenoble, France; Grenoble Institute of Neuroscience, Université Joseph Fourier, Grenoble, France Pascal Leblanc • CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, France; Institut NeuroMyoGène (INMG), CNRS UMR5310 – INSERM U1217, Université de Lyon – Université Claude Bernard, Lyon, France Yi Lee • Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Sylvain Lehmann • IRB, Hôpital St Eloi, Montpellier, France E. Levy • Department of Psychiatry, New York University Langone Medical Center, Orangeburg, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, Orangeburg, NY, USA; Division of Analytical Psychopharmacology, Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA; Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA Romain Linares • Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Pessac, France Sybren L.N. Maas • Department of Neurosurgery, University Medical Center Utrecht, Utrecht, The Netherlands; Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands Contributors
- 14. xi Imre Mäger • Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; Institute of Technology, University of Tartu, Tartu, Estonia Suresh Mathivanan • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia Kym McNicholas • Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, South Australia, Australia Michael Z. Michael • Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, South Australia, Australia; Department of Gastroenterology and Hepatology, Flinders Medical Centre, South Australia, Australia Aizea Morales-Kastresana, • National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Rienk Nieuwland • Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Joel Z. Nordin • Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden R. Perez-Gonzalez • Department of Psychiatry, New York University Langone Medical Center, Orangeburg, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, Orangeburg, NY, USA; Division of Analytical Psychopharmacology, Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA; Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA Monique Provansal • IRB, Hôpital St Eloi, Montpellier, France Graça Raposo • CNRS UMR144, Institut Curie, Paris, France Rémy Sadoul • Equipe 2, Neurodégénérescence et Plasticité, INSERM, U836, Grenoble, France; Grenoble Institute of Neuroscience, Université Joseph Fourier, Grenoble, France Mariko Saito • Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA; Department of Psychiatry, New York University Langone Medical Center, New York, NY, USA Mitsuo Saito • Division of Analytical Pshycopharmacology, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA Laurent Schaeffer • CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, France; Institut NeuroMyoGène (INMG), CNRS UMR5310 – INSERM U1217, Université de Lyon – Université Claude Bernard, Lyon, France R.M. Schiffelers • Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands Mitch Shambrook • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia Pia Siljander • Division of Biochemistry and Biotechnology, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Richard J. Simpson • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia Sisareuth Tan • Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Pessac, France Pieter Vader • Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; Department of Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands Contributors
- 15. xii Sami Valkonen • Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Didier Vilette • IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France Jeroen de Vrij • Erasmus Medical Center, Rotterdam, The Netherlands; Department of Neurosurgery, University Medical Center Utrecht, Utrecht, The Netherlands; Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands Matthew J.A. Wood • Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Rong Xu • Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia Yuana Yuana • Laboratory of Experimental Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands Contributors
- 16. 1 Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545, DOI 10.1007/978-1-4939-6728-5_1, © Springer Science+Business Media LLC 2017 Chapter 1 Methods to Analyze EVs Bernd Giebel and Clemens Helmbrecht Abstract Research in the field of extracellular vesicles (EVs) is challenged by the small size of the nano-sized particles. Apart from the use of transmission and scanning electron microscopy, established technical platforms to visualize, quantify, and characterize nano-sized EVs were lacking. Recently, methodologies to characterize nano-sized EVs have been developed. This chapter aims to summarize physical principles of novel and conventional technologies to be used in the EV field and to discuss advantages and limitations. Key words Nanoparticle tracking analysis, Electron microscopy, Dynamic light scattering, Flow cytometry, Extracellular vesicles, Resistive pulse sensing 1 Introduction Eukaryotic and prokaryotic cells release a variety of nano- and micron-sized membrane-containing vesicles into their extracellular environment, which are collectively referred to as extracellular ves- icles (EVs). EVs can be harvested from cell culture supernatants and from all body fluids including plasma, saliva, urine, milk, and cerebrospinal fluid [1]. Depending on their origin, different EV subtypes can be distinguished. Together with apoptotic bodies (1000–5000 nm), exosomes (70–160 nm) and microvesicles (100–1000 nm) provide the most prominent groups of EVs. Exosomes are defined as derivatives of the endosomal system and correspond to the intraluminal vesicles of multivesicular bodies (MVBs), which, upon fusion of the MVB with the plasma mem- brane, are released into the extracellular environment [2–4]. In contrast, microvesicles are directly pinched off the plasma mem- brane [3]. Even though the release of exosomes was initially reported in 1983 by detailed structural analysis using transmission electron microscopy [5], research on nano-sized EVs did not gain significant prominence until the discovery that small EVs transport small RNAs, including micro RNAs [6, 7]. Since then, the interest
- 17. 2 in EVs as mediators for intercellular signaling, biomarkers for diseases, drug delivery vehicles, or therapeutical agents has dra- matically increased [8, 9]. The research in the EV field is challenged by the small size of the nano-sized EVs. Apart from transmission and scanning elec- tron microscopy, established technical platforms to visualize, quan- tify and characterize nano-sized EVs were lacking. In 2011 the nanoparticle tracking analysis (NTA) was initially described as a useful technology to characterize nano-sized EVs [10, 11]. NTA has emerged as one of the most prominent, state-of-the-art tech- nologies in the EV field. In addition, other methods adopted from the field of nanotechnology are available, which have been or might be used for the characterization of EVs. This chapter aims to summarize physical principles of novel and conventional technolo- gies to be used in the EV field and to discuss advantages and limita- tions, which are summarized in Table 1. Table 1 Current methods for EV analysis Technique Particle size Time for measurement Limitations Advantages Cryo-TEM 1 nm … mm 1 h Sample preparation, only small amount of sample is analyzed Morphology DLS, homodyne 1 nm … 6 μm 1–2 min Polydisperse samples challenging, presence of large particles biases results Wide size range DLS, heterodyne 0.5 nm … 6 μm 1–2 min Analog homodyne DLS, but not as dominant Wide ranges of size and concentration NTA 20 nm … 1 μm 5–10 min Dilution necessary for high concentration, non-standardized method Visualization, resolution (even polydisperse samples), low concentrations FCM 300–500 nm … 10 μm 1 min Working range, pore blocking, calibration Fluorescence, biochemical information AFM 10–1000 nm 1 h Analog cryo-TEM Morphology RPS 50 nm to 10 μm, dependent on pore size 30 min Working range, pore blocking, calibration High resolution, compatible with buffers AF4 ca. 5 nm to 20 μm 1 h Sample dilution, interaction of sample with membrane Fractionation TEM transmission electron microscopy, DLS dynamic light scattering, NTA nanoparticle tacking analysis, FCM flow cytometry, AFM atomic force microscopy, RPS resistive pulse sensing, AF4 flow field flow fractionation Bernd Giebel and Clemens Helmbrecht
- 18. 3 2 EV Analysis There are a number of optical and nonoptical methods to analyze the size, quality, and concentration of nanoparticles. The maximal resolution (dA) of classical light microscopy depends on the wave- length of light (λ) and the numerical aperture (NA) of the lenses. It can be calculated according to the formula: dA NA . = × l 2 (1) High-quality lenses (e.g., oil immersion objectives) rarely reach apertures of more than 1.4. Accordingly, at a supposed wavelength of 550 nm, conventional light microscopes have difficulty resolv- ing structures less than 200 nm in size. To detect smaller struc- tures, electron microscopic techniques are required. Thus, EVs are conventionally analyzed by electron microscopy, usually via trans- mission electron microscopy (TEM) and in some cases by scanning electron microscopy (SEM) [10, 12]. New fluorescence based super-resolution microscopic techniques such as STED (stimulated emission depletion) or PALM (photoactivated localization micros- copy) and atomic/scanning force microscopy definitively allow for higher resolutions and certainly will provide important informa- tion on the nature of EVs in the near future [13–16]. For the preparation of EV samples for electron microscopy differ- ent methods can be used. Heavy metals, such as osmium tetroxide and uranyl acetate, increase the contrast of the analyzed samples. However, like aldehyde-based fixation methods, heavy metal treat- ment regularly results in the dehydration of the samples, resulting in EV shrinkage and deformation. Accordingly, EVs frequently adopt cup-shaped morphologies, which were initially considered as a characteristic feature of exosomes [12]. Upon using cryoelectron microscopic technologies lacking chemical fixation and staining procedures, native EV sizes and shapes can almost be conserved. Here, freshly prepared EVs are transferred to grids and immedi- ately are cryofixed in liquid nitrogen. As a result of the procedure, water is placed in a glass-like state without forming destructive ice crystals, thus, leaving the EV structure largely intact [17, 18]. Although the electron microscopic analyses provide important information on the EV morphology, this technology does not allow EV quantification; among others EVs do not quantitatively adhere to the grids. Methods based on the analysis of scattered light are eminently suit- able for the contact-free analysis of delicate samples such as bio- nanoparticles—EVs. In nearly every analysis device, ranging from dynamic light scattering (DLS) to fluorescent cell sorting, light 2.1 Electron Microscopy 2.2 Physical Background on Light Scattering Methods to Analyze EVs
- 19. 4 scattering is utilized to gain information about the samples in a fast and efficient way. Before describing current techniques, a brief physical background about the light scattering features of small particles should be given. When small particles (ranging in size from approximately 100 nm to several μm), such as in diluted milk or fog, are illuminated by a directed beam of light from a laser pointer, the light beam becomes visible as the particles scatter the incident light. Single, larger par- ticles can even be recognized by eye, like dust in the sunlight. In the middle of the nineteenth century, Tyndall (1820–1893) observed this phenomenon and used it for the detection of small particles in liquids. Although he probably was not the first who discovered this phenomenon, the effect has been termed the “Tyndall effect.” The scattered light contains information, allow- ing detection and analysis of the particles which is employed in common and new techniques based on light scattering. Bearing in mind the principle of energy conservation, energy can- not be created nor destroyed but only changed from one form into another. As an example, energy (the momentum) can be trans- ferred from one billiard ball to another. In the elastic case, the bil- liard balls deform during collision (although this cannot be seen by eye), kinetic energy is transferred and the billiard balls return to the original form. In contrast, if one of the balls would be made of modeling mass, parts of the transferred energy lead to inelastic deformation of the modeling mass ball and only parts of the momentum are transferred as kinetic energy. This example reflects the underlying principle in light scatter- ing. Light is an electromagnetic wave with wavelengths visible to the human eye ranging from 380 to 780 nm. The electromagnetic wave consists of a large number of small discrete energy packages, the photons. The energy of a photon, transferring the energy (E), can be calculated via the expression E h c = 0 l (2) (h Planck’s constant, h=6.626 x 10−34 Js; c0: speed of light in vac- uum, c0 =2.998×108 ms−1 ). Upon illumination of a given particle, the light wave inter- acts with the particle; more precisely, the photons of the light wave transfer their energy to the particle’s electrons. As a con- sequence the electrons oscillate and finally energy can be released in form of scatter light in all directions uniformly. In elastic scattering, the energy transferred by the photons is identical to the energy of the scatter light; consequently, the incident and scatter light have identical wavelengths (Fig. 1). 2.2.1 Tyndall Effect 2.2.2 Elastic and Inelastic Light Scattering Bernd Giebel and Clemens Helmbrecht
- 20. 5 Examples for elastic light scattering are Rayleigh scattering and Mie scattering, which will be explained below. If the energy of the incident and scatter light differ from each other, the pro- cess is termed inelastic light scattering. Light scattered in a Raman process is an example of inelastic light scattering. In such a process a part of the energy of the incident light is trans- formed into another form of energy, e.g., heat or vibrational energy. In Stokes Raman scattering, the wavelength of the scat- tered light is longer than the incident light. In anti-Stokes Raman scattering the wavelength of the scattered light is shorter than the wavelength of the incident light. The additional energy derives from vibrational energy of the molecules of the particle, e.g., when the molecules are in excited state. Of note, compared to elastic scattering, Raman scattering is very weak and requires well thought-out arrangements for detection [19, 20]. The characteristic of how particles scatter light is mainly related to their size. Within the scope of this chapter, we focus on Rayleigh and Mie scattering. Rayleigh scattering describes the elastic scattering of electro- magnetic waves on particles with sizes rather small compared to the incident wavelength r 0.2 λ. The intensity of the scattered light is inversely related to the fourth power of the wavelength λ of the incident light. Consequently, light with shorter wave- lengths is scattered with higher intensities than light with lon- ger wavelengths. A well-known phenomenon which can be explained by Rayleigh scattering is the blue color of the sky; 2.2.3 The Influence of Particle Size 2.2.4 Rayleigh Scattering Fig. 1 Incident light wave with Ei and λi shifts the electrons of the particle from a ground state E0 to a virtual level E1. Elastic scattering: from the virtual level E1, electrons return to the ground state, no energy is transformed (e.g., Rayleigh scattering). Inelastic scattering: electrons do not return to the ground state E0. Parts of the incident energy (Eem″) are transformed into other energy forms (e.g., Stokes scattering) Methods to Analyze EVs
- 21. 6 molecules in the atmosphere scatter the blue parts of sunlight approximately ten times stronger than the red parts. The scattering intensity I also depends on the index of refraction n of both, of the particle (n1) and surrounding medium (n2). The refractive index is defined as the ratio between the speed of light in a given material and in a vacuum. The relative refraction index m=n1/n2; n1 and n2 are the refractive indices of particle and surrounding media, respectively. Considering all these parameters, the intensity (I) of the Rayleigh scattering at a certain distance (R) and scattering angle (θ) [21] is given by: I I R m m d = ´ + æ è ç ö ø ÷ - + æ è ç ö ø ÷ æ è ç ö ø ÷ 0 2 2 4 2 2 2 6 1 2 2 1 2 2 cos q p l (3) Of note, the intensity of Rayleigh scattering is proportional to the sixth power of the size of small particles, which restricts the size detection limit of many scatter based methods. In contrast, the irradiation intensity (I0) is only linearly linked to the intensity of Rayleigh scattering. A large difference in the refractive index of the surrounding medium and the illuminated particles (e.g., water n2 =1.333) increased the intensity of the scattered light. Particles with similar or larger sizes than the wavelength of the incident light cause Mie scattering. The formula to calculate the intensity of Mie scattering at a given angle and distance of larger particles is much more complex and is neglected here. Particles with an approximate size of the wavelength of the incident light can be considered as an aggregation of material, whose oscillating electrons influence each other and may scatter the light toward a certain direction. As a consequence, the Mie scattering intensity is less dependent on the wavelength of light than Rayleigh scattering. For example, waterdrops in clouds cause wavelength-independent Mie scattering; that is the reason why clouds appear white. For a more detailed description on light scattering, we like to refer to more specific literature [22, 23]. 3 Methods Based on Light Scattering An advanced technology applying the scattering light for the characterization of nanoparticles is the method of dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS). Here, a distinct proportion of the sample volume—regu- larly a few microliters—is illuminated with a laser beam. The light scattered from the particles within the illuminated part of the probe is recorded over time [24]. Due to their Brownian motion, the particles in the sample are constantly moving, some of them leaving and some of them entering the illuminated part of the 2.2.5 Mie Scattering 3.1 Dynamic Light Scattering (DLS) Bernd Giebel and Clemens Helmbrecht
- 22. 7 probe. This causes fluctuations of the scattering light, which is registered by the detector. Since smaller particles move faster within the probe than larger particles, smaller particles cause higher fluctuations than larger particles. By the combination of mathematical models of the Brownian motion and the light scat- tering theory differential particle sizes can be calculated within seconds [25]. While in the beginning of commercial DLS (around 1970) only narrow size distributions could be measured, the range of modern DLS instruments typically covers sizes ranging between 1 nm and 6 μm [26]. To obtain optimal results, the presence of contaminants such as dust particles, air bubbles, debris and inorganic particles, which can derive from laboratory water (e.g., silicates, phosphates, carbonates), must be circum- vented. For better reproducibility, optimized sample preparation including filtration of buffers is mandatory [27]. Depending on the position of the detector, two different DLS systems are commercially available, the homodyne and the heterodyne DLS. In a homodyne DLS setup, the laser and detector are arranged perpendicular to each other (Fig. 2). The incident light with the intensity I0 illuminates the sample and becomes partially scattered by the particles suspended in the probe. The intensity of the scattered light (IS) is recorded by the detector. Critical parameters in this setting are the distance the light has to pass through the sample until it reaches the detector and the concen- tration of the particles. If the particles are too concentrated, sec- ondary scattering occurs diminishing the amount of scatter light that reaches the detector. Hence, appropriate dilutions have to be titrated to obtain valid data [28]. Within heterodyne DLS systems the backscattered light is analyzed (Fig. 2). The incident laser light is coupled into an opti- cal fiber to illuminate the probe with the intensity I0. Only light, which is scattered by the particles within the probe in an angle of 180°, can reenter the optical fiber and become transmitted with Fig. 2 Principle of homodyne and heterodyne DLS systems Methods to Analyze EVs
- 23. 8 an intensity IS to the detector [29]. In addition to the average size distribution of the particles in the probe and following calibration, heterodyne DLS regularly enables to determine the particle con- centration of given probes. For appropriate measurements of par- ticle sizes, analyses of polydisperse probes require particle size differences with ratios of d1/d2 1.8 [30]. Regularly, a 20–50 μL sample volume is sufficient to determine the average particle size distribution on commercial DLS instru- ments in less than a minute. Analyses of monodisperse samples, i.e., samples only containing particles with the same size, yield reli- able results. In the case of polydisperse samples such as blood plasma samples, the results may be less clear and require knowl- edge of the applicable mathematical model. The results are dis- torted by larger particles with diameters in the micrometer range, already when they are present at low concentrations [30]. Upon analyzing samples with high particle concentrations or samples containing larger agglomerates, heterodyne DLS instruments pro- vide more flexibility than homodyne instruments, but still are lim- ited compared to other techniques such as the nanoparticle tracking analysis (NTA) [31]. In 2011 NTA was reported to provide a suitable method for EV characterization for the first time [10, 11]. Since then, NTA has emerged as one of the standard techniques for the characterization of EVs. It also allows analyses of larger particles within the microm- eter range and thus has also been designated as particle tracking analysis (PTA). Analogous to DLS, NTA records the Brownian motion of small particles. Similar to DLS, particles in the sample are visual- ized by the illumination with incident laser light. The scattered light of the particles is recorded with a light-sensitive CCD cam- era, which is arranged at a 90° angle to the irradiation plane (Fig. 3). The 90° arrangement, also known as ultramicroscopy, allows detection and tracking of the Brownian motion of 10–1000-nm-sized vesicles. Using a special algorithm the size of each individually tracked particle is calculated, thus simultane- ously allowing determination of the average size distribution of particles in a given sample as well as their concentration. Even though the NTA technology is relatively new on the market, it originated almost 25 years ago [32]; the commercial implementa- tion of this technique required the availability of fast computer systems that are able to cope with the computationally intensive video analysis in reasonable time frames. A brief introduction of the physical principle underlying NTA is as follows: When small particles are dispersed in a liquid (the so- called continuous phase, e.g., water), the particles move randomly in all directions. This phenomenon is termed diffusion and is expressed by the diffusion coefficient (D). In more detail, the 3.2 Nanoparticle Tracking Analysis (NTA) Bernd Giebel and Clemens Helmbrecht
- 24. 9 undirected migration of given particles is caused by energy trans- fers from surrounding water molecules to the particle. In the absence of any concentration gradient within the dispersion and upon long-term observation, the distances small particles move in any direction should neutralize each other over time, leaving a total movement of almost zero. However, during given time inter- vals, diffusing particles move within certain volume elements. In NTA the time t between two observation spots is quite short (~30 ms). The distance particles have moved during the time inter- val are recorded and quantified as the mean square displacement (x2 ). Depending on the number of dimensions (one, two or all three dimensions) the diffusion coefficient can be calculated from the mean square displacement as follows: D x t D x y t D x y z t = = = 2 2 2 2 4 6 , , , . Via the Stokes-Einstein relationship, the particle diameter d can be calculated as function of the diffusion coefficient D at a tempera- ture T and a viscosity η of the liquid (kB Boltzmann’s constant) [33]: D k T d = 4 3 B . ph In NTA, the particle fluctuation of a single particle is registered in two dimensions. After combining the Stokes-Einstein relationship and the two-dimensional mean square displacement, the equation can be solved for the particle diameter d with: d k T t t x y k T x y = × = 4 3 4 16 3 2 2 B B . ph ph , , Fig. 3 Schematic setup of a nanoparticle tracking analyzer Methods to Analyze EVs
- 25. 10 By simultaneously tracking several particles, their diameters can be determined in parallel. Figure 4 shows a typical particle size distri- bution of vesicles harvested from blood plasma. The lower limit of the working range, i.e., the smallest detect- able particle size, depends on the scattered intensity of the particle (compare Eq. 3), the efficiency of the magnifying optics and the sensitivity of the camera [34]. Silver and gold nanoparticles are strong scatterers due to the comparably large refractive indices of 2–4 and can be detected down to sizes of ~10 nm. Biological nanoparticles such as EVs have refractive indices of around 1.37– 1.45 resulting in a limit of detection of 30–50 nm for NTA [35]. NTA allows the direct measurement of concentration as single particles in the illuminated volume are visualized. Thus, NTA is an absolute measurement technique allowing the determination of total surface or volumes of particles in a sample (see Fig. 4). For the measurement of concentration, the instrument is calibrated with Fig. 4 Particle size distributions of vesicles in blood plasma. The particle size distributions range from 100 to 1000 nm dependent on weighing according to number, area, or volume. NTA as absolute technique allows quantification of concentration, area, and volume of vesicles present in the sample Bernd Giebel and Clemens Helmbrecht
- 26. 11 size standards of known size and concentration. The visualization of the sample gives a unique impression on the quality of the sam- ple, such as the presence of agglomerates. The working range of 0.5×106 and 1×1010 particles per cm3 is very low compared to DLS, allowing NTA to analyze low concentrated samples. To record representative size distribution profiles, it is recommended to analyze a range of 1000–10,000 single particles. While in the early stages of NTA development, the manual adjustment of microscope and laser was time-consuming, nowa- days, the measurement cell is aligned within minutes. Currently, commercial NTA instruments are offered by only two companies (Malvern Instruments Ltd. and Particle Metrix GmbH). Depending on the model temperature control, conductivity and zeta potential measurement are integrated. The zeta potential reflects the surface charge of given particles, which might be related to their stability. Currently, efforts are undertaken to implement additional components, which, for example, can auto- matically dilute probes to optimal particle concentrations, record electrochemical parameters (e.g., the pH of the probe), and allow for the specific characterization of fluorescent-labeled EVs. The quality of an NTA result is influenced by particle con- tamination. In addition to the contaminating particles, which were mentioned in the section of DLS, high concentrations of stabilizing agents (e.g., surfactants) are critical as soon as they reach their critical micellar concentration (CMC). Contaminating particles may derive from diluents (distilled water or buffer agents) or from chemicals used during preparation of samples. Regularly, chemicals are not certified for the absence of nanoparticles. Precipitates of phosphates, carbonates, or silicates as well as dust can be removed by filtration of the buffers, ideally with pore sizes below 50 nm. Degassing in ultrasonic bath is also helpful to remove air bubbles [34]. For the characterization of EVs, it would be desirable to simul- taneously analyze the presence of different molecules expressed on the surface of EVs using a high-throughput technology. At the cellular level, such analyses are regularly performed by FC. However, due to the configuration of conventional flow cytometers, the size detection limits of particles lie somewhere between 300 and 500 nm [36]. Thus, by means of conventional flow cytometry, only large EVs can be analyzed at an individual particle level. To this end, EV FC analyses have indeed already been carried out on larger EVs, particularly in the area of plate- let research. In the literature corresponding EVs are usually referred to as microparticles [37–39]. Analyses of smaller EVs by flow cytometry require either a special mechanical setup, or EVs must be bound by immunological methods to carrier particles. 3.3 Flow Cytometry (FC) Methods to Analyze EVs
- 27. 12 Magnetic carrier particles or latex beads can be coated with antibodies that recognize epitopes on EVs, e.g., anti-CD63 anti- bodies. If the antibody-coated beads are added to EV-containing samples, aggregates between the beads and the EVs are formed, which can be concentrated by magnetic separation or by low-speed centrifugation, respectively. For an appropriate aggregation, suffi- cient quantities of EVs need to be present in the sample; the beads should get saturated with EVs, otherwise aggregates with several beads might form. The aggregate formation of EVs with several beads can be reduced by vortexing or pipetting. In analogy to cells, the formed bead-EV aggregates can be labeled with different fluorescence- labeled antibodies. Due to the presence of the beads, these aggregates are big enough to be analyzed on conventional flow cytometers [40–43]. This technology offers the great advan- tage for a fast and comprehensive EV characterization. However, since only aggregates and not individual EVs are analyzed, this form of analysis is a bulk analysis and finally may not reveal much more information than conventional Western blots. Irrespective of the low size resolution of conventional flow cytometers, analyses of small EVs at the single-particle level pro- vide several challenges. As long as the particles are larger than the wavelength of light, their size corresponds to the amount of the forward-scattered light, which is measured at the forward scatter detector. If the particle sizes are around or below the wavelength of the light, the intensity of light scattered to the side increases proportionally to the forward-scattered light. Accordingly, the size of particles that are smaller than the wavelength of the incident light can better be determined upon measuring the scattered light at the side scatter detector than on the forward scatter detector. Alternatively, an extended forward scatter detector can be used, which collects the forward-scattered light and proportions of the side scattered light. Groups that have optimized the setup of configurable flow cytometers for the measurement of nano-sized particles were already able to analyze viruses and EVs at a single-particle resolution [44–46]. Essential prerequisites for such measurements are the reduction of signal-to-noise ratio and an increase in the sensitivity of the scatter light detection. According to the formula of the Rayleigh scattering, a linear increase in sensitivity can be achieved by increasing the intensity of the laser light [44]. In addition, the signal-to-noise ratio largely depends on the processing of the sheath fluid. Regularly, commercial products are sterilized by filtra- tion through 0.22 μm filters, which is not sufficient to remove background noise producing nanoparticles such as calcium phos- phate or calcium carbonate nanoparticles. Thus, filtration through 0.05 μm filters is highly recommendable [44]. The background noise can also be reduced upon staining EVs with a strong fluores- cent dye, e.g., the membrane-intercalating PKH67, and by trig- Bernd Giebel and Clemens Helmbrecht
- 28. 13 gering the subsequent flow cytometric measurements on the fluorescence and not as conventionally on the scattered light [46]. The disadvantage here is that aggregates of the unbound fluoro- chromes should be removed before stained EVs get analyzed. Even though it is time consuming, currently, density gradient centrifu- gation appears as the most appropriate technology to separate fluo- rochrome aggregates and stained EVs. Irrespectively of this, EVs can also be marked with fluorescence conjugated antibodies allow- ing for the specific analyses of antigens of interest [46, 47]. Since the surface of EVs is orders of magnitude smaller than that of cells, antibodies should be used being conjugated to very bright fluoro- chromes such as B-phycoerythrin (B- PE) or R-PE. Usage of anti- bodies with weaker fluorochromes can only be recommended, when corresponding epitopes are known to be expressed on the EVs very abundantly [47]. Another challenge is the concentration of the EVs to be mea- sured. Ideally, for single particle analyses, the concentration of par- ticles to be measured should be in the range of 5×105 to 5×106 particles per ml sample liquid. If particles are higher concentrated, swarm detection can occur, that is, the simultaneous detection of several particles at a given moment [48]. Following enrichment of EVs, the concentration regularly strongly exceeds this value; con- sequently, probes to be measured have to be diluted to sometimes homeopathic appearing dilutions. Raman scattering is a form of inelastic light scattering [19]. Even though most of the incident light is scattered in an elastic manner, each molecule also specifically scatters light in an inelastic manner and thus generates individual Raman spectra of the scattered light. Raman microspectroscopy allows the recording and analysis of sample spec- tra and thus gives information on molecular composition of probes of interest. This technique has been used to analyze the composition of EVs and allowed discrimination of different EV subtypes from each other [49]. Especially when combined with atomic force microscopy, Raman spectroscopy might offer a very potent technology to analyze and discriminate different EV subtypes [50]. Raman microspectroscopy is a relatively high-priced and spe- cialized technique. Setup and acquisition require a relatively large amount of time, resulting in an incompatibility with high- throughput analyses (10–100 vesicles per hour). Due to the low intensity of the Raman scattering signal (approx. 1:10,000 of elastic scattering), the measurement is influenced by artifacts demanding high grade of manual effort and expertise of the oper- ating personnel. During measurement, EVs are exposed to a high- intensity light beam, which can induce photostress and cause adverse effects. Depending on the dose and wavelength of the incident light beam, (photo) reactions might be induced in the EVs and change them irreversibly [49]. 3.4 Raman Microspectroscopy (RM) Methods to Analyze EVs
- 29. 14 In the 1980s, considerable efforts were made to develop tech- niques allowing resolving solid state surfaces at atomic levels. As a result, the atomic force microscope (AFM) [51] and later the scan- ning tunneling microscope (STM) were developed. AFM is based on a tip mounted on a cantilever that is moved like the pick-up of a record player in a defined distance over the surface of the material to be analyzed. The radius of the tip ideally is reduced to that of a few atoms. The torsion of the cantilever is a measure for the forces between tip and surface as function of the distance. The tip is either attracted (e.g., van der Waals forces) or repelled (e.g., electro- static forces) from the surface resulting in characteristic force-distance curves. In the beginning, AFM has been utilized for the quantitative description of the topology of solid-state surfaces under vacuum con- ditions. Meanwhile immobilized particles such as vesicles can also be analyzed in buffers [36, 52, 53]. Thus, AFM became a feasible method for the characterization of EVs, especially to analyze their size and topology [54, 55]. However, as immobilization of EVs might affect their topology, results are influenced by the mode of sample preparation [56]. Resistive pulse sensing (RPS) is a technology to measure absolute sizes and the concentration of particles in suspension, whose sizes range from 100 nm to 100 μm. In principal, the system contains two cells, both equipped with an electrode. The cells are con- nected by membrane containing a small pore or a micro-channel, regularly with pore sizes below 1 μm (Fig. 5). To analyze the particle concentration and the average size distributions of sus- pensions, an electric field is applied onto the electrodes. As a con- sequence, charged particles migrate to the anode or cathode, respectively. In analogy to the Coulter principle, each time a par- ticle passes through the pore, the electrical resistance of the buf- fer gets altered. These alterations in resistance are recorded. Since alterations in the resistance depend on the volume of the migrat- ing particles, the particle sizes and their zeta potential can be calculated [57]. As a prerequisite for this method, the pore diam- eter (q) has to be much smaller than the pore thickness (l). Following calibration with particles of defined sizes, particle sizes and their zeta potentials can be calculated; they are proportional to the shapes and heights of the recorded pulses. Considering the pore of the membrane as a cylinder, the electrical resistance (R) of the pure buffer can be calculated as: R l A = r ρ: specific resistance of the buffer, l: pore thickness (typically several tens of μm), A: pore area. 3.5 Scattered-Light- Independent Technologies 3.5.1 Atomic Force Microscopy (AFM) 3.5.2 Resistive Pulse Sensing (RPS) Bernd Giebel and Clemens Helmbrecht
- 30. 15 With A=π/4×q2 the pore area is related to the pore diameter (q). In reality, each particle contains a specific electrical resistance which theoretically has to be considered. However, specific electrical resistances of given particles are high. If particles are considered as insulators, their specific electrical resistance can be neglected [58]. Provided the platform is equilibrated with particles of known concentration, the estimated count rates of given particle suspen- sions to be analyzed reveal their particle concentrations. The upper end of the working range is limited by the pore size, the lower end on the sensitivity in the detection of resistance changes (typically ~0.2 q). Before usage, every membrane needs to be cali- brated with size standards. Upon analyzing biological samples, pore blocking often increases the analysis time per sample of up to 1 h. RPS instruments capable of detecting particles in the lower nanome- ter ranges (in general 100 nm) are under development [59]. The family of field flow fractionation techniques (FFF) comprises instruments separating polydisperse samples in individual fractions while simultaneously determining their particle size. FFF techniques are characterized by high resolution and compatibility to flow detec- tors and have already been used to characterize EVs [60, 61]. The separation is based on the so-called cross-flow principle, in which two orthogonal forces act on the particle. Depending on the underlying FFF technique, the forces can be created differ- ently, either by friction in flow field FFF (FFFF, F4 ), sedimentation (sedimentation FFF, SdFFF), or an electrical field (ElFFF). FFFF and SdFFF are the most common techniques [62]. In FFFF a separation channel with an asymmetrical flow pro- file (asymmetrical FFFF, AF4 ) is prevailingly used; it gives the most reproducible results with lowest sample loss (Fig. 6). Before 3.5.3 Field Flow Fractionation (FFF) Fig. 5 Resistive pulse sensing (RPS). Left: Typical setup with two cells separated via an insulating membrane with a single pore. Right: Transient signal of current representing a (1) large-, (2) small-, and (3) medium-sized particle. Following calibration, the count rate, i.e., the number of pulses per time interval, reflects the concentration of the particle suspension to be analyzed Methods to Analyze EVs
- 31. 16 fractionation starts, the sample is focused as a small band on the semipermeable membrane. During fractionation two perpendicu- lar flows act on the sample, a laminar flow and a cross flow. The cross flow counteracts the diffusion tendency of the particles away from the membrane. Approximately 10 min after starting the measurement, the diffusion and cross flow are equilibrated and the particles have accumulated in a certain distance to the membrane, which depends on the diffusion coefficient of the par- ticles. Since smaller particles contain higher diffusion coefficients than larger ones, smaller particles accumulate at higher distances away from the membrane than larger particles. The laminar flow transports particles to the detector. The closer particles accumu- late toward the middle of the flow channel, the faster they are transported to the detector. As a result, smaller particles arrive earlier at the detector than larger ones. For the detection a single detector or a combination of detectors can be used. The follow- ing detectors are available: absorption detector (diode array), fluorescence detector, scattering light detector, and atom spec- troscopic detector (e.g., inductively coupled plasma mass spec- trometer, ICP-MS). The usage of detectors allowing deciphering the chemical composition of probes (HPLC, Raman, TXRF) has been reported [63]. Typically, polydisperse samples containing three or more dif- ferent components can be separated. The injection volumes depend on the type of sample and the concentration of its particles; it may vary from between 20 μl to 2 ml. To set up a successful separation composition, concentration and pH of eluents need to be optimized to prevent aggregation or irreversible binding of the particles to the membrane [64]. During separation the particles are regularly diluted 100- to 1000-fold. Fig. 6 Asymmetrical flow field flow fractionation (AF4 ). Before separation, given samples are concentrated on the membrane. Depending on their sizes, particles diffuse from the membrane and accumulate at certain posi- tions in which equilibriums of diffusing and cross flow forces are given. Simultaneously to their diffusion, the laminar flow transports the particles toward the detector at the end of the flow channel Bernd Giebel and Clemens Helmbrecht
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- 36. 21 Chapter 2 Tunable Resistive Pulse Sensing for the Characterization of Extracellular Vesicles Sybren L.N. Maas, Marike L.D. Broekman, and Jeroen de Vrij Abstract Accurate characterization of extracellular vesicles (EVs), including exosomes and microvesicles, is essential to obtain further knowledge on the biological relevance of EVs. Tunable resistive pulse sensing (tRPS) has shown promise as a method for single particle-based quantification and size profiling of EVs. Here, we describe the technical background of tRPS and its applications for EV characterization. Besides the stan- dard protocol, we describe an alternative protocol, in which samples are spiked with polystyrene beads of known size and concentration. This alternative protocol can be used to overcome some of the challenges of direct EV characterization in biological fluids. Key words Extracellular vesicles, Exosomes, Microvesicles, Characterization, Quantification, Size distribution, qNano, Resistive pulse sensing 1 Introduction Due to their small size (50–1000 nm), accurate characterization of extracellular vesicles (EVs) is technically challenging. Over time, different techniques have been developed to overcome these chal- lenges. Most of these techniques are based on bulk analysis of EVs. For instance by total protein quantification, western blotting, bead-based flow cytometry [1] or modified protein microarrays [2]. However, alternative techniques, that allow for single particle analysis of EVs, have become recently available [3–8]. One of those techniques, provided by the qNano platform (Izon Science Ltd), is tunable resistive pulse sensing (tRPS) (Fig. 1). In tRPS, a non-conductive membrane (“nanopore”) separates two fluid cells [9] (Fig. 2). This nanopore is punctured to create a single conical shaped opening (Fig. 2, top-left). Once a voltage is applied, a current of charged ions through the nanopore is estab- lished. This baseline current is distorted, as observed by the appear- ance of peaks or “pulses,” as particles move through the nanopore (Fig. 2, bottom). Once a particle enters the sensing zone of the Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545, DOI 10.1007/978-1-4939-6728-5_2, © Springer Science+Business Media LLC 2017
- 37. 22 Fig. 1 Photographs of the qNano instrument and instrument parts 1 2 3 1 2 3 - + V Fig. 2 The working mechanism of tunable resistive pulse sensing (tRPS). A membrane (“nanopore”) with a nanosized, stretchable pore is separating two fluid compartments (top-left).After applying a voltage across the nanopore, a baseline current is established (bottom) which is disrupted by the movement of particles through the nanopore. As a particle moves towards the opening (timing 1), it starts to reduce the flow of ions through the nanopore (top-right) which will be maximum as the particle enters the nanopore opening (timing 2). This disruption reduces as the particle moves across and exits the nanopore (timing 3) Sybren L.N. Maas et al.
- 38. 23 nanopore [10] (Fig. 2, timing 1), the flow of charged ions, and thus the baseline current, will be altered (Fig. 2, top-right). As the par- ticle enters the conical opening, the relative blockade of the baseline current will be maximum (Fig. 2, timing 2). This blockade will gradually decrease to baseline levels as the particle moves further through the nanopore (Fig. 2, timing 3). To characterize particles in a sample, a calibration sample of (polystyrene) beads of known volume and concentration is measured first. The magnitude of pulses and the particle rate induced by this reference sample can subsequently be used to calculate the size profile and concentration of the particles in the measurement sample [11, 12]. The movement of particles through the nanopore is based on several independent forces, being electrokinetic (electrophoretic and electro-osmotic) and fluidic forces [10]. The variable pres- sure module (VPM) can be used to apply additional external force and should be used (≥0.8 kPa) to minimize interfering electrokinetic forces when analyzing particles using the smaller (NP100-NP200) nanopores [13]. Characterization of EVs using tRPS is technically challenging. Due to the heterogeneous nature of EVs a large size range of parti- cles is usually present in a sample. Larger-sized EVs may clog the nanopore, thereby obstructing the measurement. Secondly, the sample with calibration beads should consist of the same buffer com- ponents as the EV sample. This may be technically unfeasible, as the buffer components are regularly unknown when measuring EVs, especially when measuring EVs directly in a biological sample. This problem can be overcome by using a “spiking” approach, in which the calibration beads are added to the measurement sample [3]. Here, we describe two different approaches for the characteriza- tion of EVs using tRPS. First, the standard protocol is described, which often suffices for the characterization of purified EVs. Secondly, we describe the alternative spiking approach, which could be of benefit when characterizing EVs in biological samples. 2 Materials 1. qNano instrument (Izon Science Ltd, Christchurch, New Zealand). 2. Variable Pressure Module (Izon Science Ltd, Christchurch, New Zealand). 3. Polystyrene calibration particles (Izon Science Ltd, Christchurch, New Zealand) (see Note 1). 4. Nanopores (Izon Science Ltd, Christchurch, New Zealand) (see Note 2). 2.1 qNano Specific Equipment/Materials tRPS for EV Characterization
- 39. 24 1. Filter-tip pipette tips (see Note 3). 2. Sonication bath (see Note 4). 3. Lint-free tissues (see Note 5). 4. Phosphate buffered saline (PBS). 5. Digital calipers (supplied with the qNano instrument). 1. Izon Control Suite (Izon Science Ltd, Christchurch, New Zealand). 2. Spreadsheet software (see Note 6). 3 Methods The standard protocol of tRPS-based EV quantification involves separate measurement of a (polystyrene bead-containing) calibration sample and the EV-containing sample. 1. Connect the qNano instrument to a computer running the Izon Control Suite Software. Make sure no sources of elec- trical interference are located close to the instrument (see Note 7). 2. Wet the lower fluid cell by introducing 75 μl PBS and immedi- ately removing it again (see Note 8). 3. Place the nanopore of choice (see Note 2). To calibrate the stretch, use the digital calipers to measure the distance between two opposing arms of the qNano. 4. Stretch the nanopore to 47 mm and reapply 75 μl to the lower fluid cell. Prevent the formation of air bubbles in the lower fluid cell. If air bubbles are formed, remove and reapply the PBS. 5. Place the upper fluid cell and the shielding cap (which creates a “Faraday cage”) on the nanopore. Add 40 μl PBS into the upper fluid cell and apply a voltage. Make sure a stable baseline current is established (see Note 9). 6. Dilute the calibration particles in PBS to the target concentra- tion of the used nanopore (see Note 10). 7. Remove the PBS from the upper fluid cell and apply 40 μl of the calibration particles into the upper fluid cell. Make sure a stable baseline current is established (see Note 9). Reduce the applied stretch slowly towards 43 mm and observe the block- ades caused by the calibration particles. Stop reducing the stretch when the mode blockade caused is at least 0.1 nA, but preferable 0.3 nA (see Notes 11 and 12). 2.2 General Laboratory Equipment/Materials 2.3 Software for Data Recording and Analysis 3.1 Standard Protocol Sybren L.N. Maas et al.
- 40. 25 8. Apply ≥0.8 kPa pressure using the VPM and click “record” (see Note 13). Make sure that a particle rate (see Note 14) of 100 min− and a mode blockade height of 0.1 nA is recorded (see Note 12). 9. If the baseline current suddenly drops or keeps drifting during recording, pause the recording and try to reestablish a stable current (see Note 9). 10. Record 500 particles, for at least 30 s (see Note 14). Fill out the details of the calibration sample in the pop-up form. 11. Optionally, multi-pressure measurement can be performed (see Notes 13 and 15). Hereto, add at least 0.2 kPa and record a second measurement (more steps could increase accuracy). 12. Remove the calibration sample and wash the upper fluid cell by resuspending 100 μl PBS in the upper fluid cell 3–4 times. Remove residual PBS by usage of the lint-free tissue (see Note 16). 13. Introduce the EV sample and make sure the baseline current is within 3% of the baseline for the calibration sample (see Note 17). 14. Record the sample at the same VPM pressure(s) as applied for the calibration sample. 15. Click the “Analyse data” tab and right-click on “Unprocessed files” and select “Process files”. 16. Click on the checkbox in the “calibrated” column next to one of the sample files. This will initialize the calibration pop-up menu. Select the “multi-pressure measurement” tab if appli- cable and select the sample files and calibration file(s). 17. Once calibrated, an EV sample file will display a size distribu- tion in nm instead of nA (Fig. 3, right). Click on “Preview” to generate a .pdf file containing statistics such as the concentra- tion (measured and raw if a diluted sample was used). The standard protocol for tRPS-based EV quantification relies on usage of appropriately formulated calibration samples (i.e., with the diluents resembling the fluid of the EV sample). This may be unfea- sible for biological fluids, since their exact composition may be unknown rendering their simulation impossible. Secondly, the vol- ume of the biological sample (e.g., only 100 μl of plasma) may be insufficient for preparation of calibration fluid (which usually can be done by removal of small particulate matter by ultracentrifugation or filtering). In such cases, an alternative is provided by performing a spiking protocol, in which calibration beads are introduced in the EV sample [3]. This methodology can also be used when samples are measured over a prolonged period of time and stable nanopore conditions cannot be guaranteed due to nanopore clogging. 3.2 Spiking the Sample with Polystyrene Beads of Known Size and Concentration tRPS for EV Characterization
- 42. 27 1. Setup the qNano instrument as outlined in Subheading 3.1 steps 1–5. 2. Check the approximate particle rate of the EV samples. 3. Dilute the EV sample in PBS (see Note 18). 4. Determine the dilution of polystyrene beads that is needed to obtain a count rate that resembles the count rate of the EV samples (see Note 19), and check for the ability to distinguish EVs and polystyrene beads (see Note 20). 5. Prepare the samples by diluting polystyrene beads into the samples (see Note 21). Also prepare a “beads-only” sample (see Note 22). 6. Record the beads-only and sample measurements, preferable in triplicate (see Note 23). 7. Process all files as outlined in Subheading 3.1 step 15. 8. Display the size distribution graphs (uncalibrated) of the beads-only samples and sample files (Fig. 4, left). Determine at which nA value a cutoff can be set to distinguish the two popu- lations (Fig. 4) (see Note 24). 9. Obtain the total particle count (in sample details window) for each sample and put this into a spreadsheet software program (Table 1). 10. Click the “filter options” button to obtain the filter settings. Enter the cutoff obtained in step 8 and filter the samples. Make sure to select the “apply to all samples in group” checkbox to filter all samples directly. 0 1 2 3 4 5 6 7 8 9 % Population (by count) % Population (by count) 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 2,2 Blockade Height (nA) Cell culture supernatant replicate #1 Cell culture supernatant replicate #2 Cell culture supernatant replicate #3 0 100 200 300 400 500 Particle count 0 40 80 120 160 200240 280 Time (s) 0 2 4 6 8 10 12 14 16 18 0 40 80 120 160 200 240 280 320 Particle Diameter (nm) Cell culture supernatant replicate #1 Fig. 4 Quantification and size estimation of EVs by spiking the sample with polystyrene beads of known size and concentration. Three replicates of glioblastoma cell culture supernatant spiked with 203 nm polystyrene beads are measured (left). All particles smaller than 0.48 nA were determined EVs. The EV-to-beads ratio is used to calculate the concentration of EVs. The spiked polystyrene beads can be used to obtain an accurate size distribution without the need of an external calibration sample (right) tRPS for EV Characterization
- 43. 28 11. Obtain the total particle counts for each sample after the filter step. Fill out these numbers into the spreadsheet software (see Table 1 for an example calculation). 12. Subtract the EV counts from the total counts to obtain the amount of calibration particles. Subsequently, divide the num- ber of EVs by the number of polystyrene particles to obtain the EV-to-bead ratio. 13. To account for background particles, subtract the average ratio obtained for the beads-only samples from each EV-to- bead ratio. 14. Multiply the EV-to-bead ratio to the concentration of polysty- rene beads in the sample. Secondly, multiply this value by the dilution factor of the EVs (see Note 25) to obtain the raw concentration of EVs. 15. Optionally: introduce a correction when overlap of EVs and polystyrene beads is observed (see Note 26). The above-described spiking procedure can also be utilized to obtain a proper size distribution profile of EVs in case the prepara- tion of appropriate calibration samples is impossible. 1. Prepare, measure, and process the EV samples as outlined in Subheading 3.2 steps 1–8. 2. Once processed open the sample of interest twice in the Izon Control Suite. 3.3 Obtaining an EV Size Distribution from a Spiked Sample Table 1 Example calculation of EV concentration using the alternative spiking method Sample Beads-only #1 Beads-only #2 Replicate #1 Replicate #2 Replicate #3 Average current 63 60 62 61 61 Rate 57 74 112 133 105 Cutoff used 0.48 0.48 0.48 0.48 0.48 Total particles 297 299 502 495 500 Extracellular vesicles (EVs) 27 46 254 240 246 Beads+multimers 270 253 248 255 254 EVs/beads 0.100 0.182 1.024 0.941 0.969 Sample—background 0.88 0.80 0.83 EVs (108 /ml) in sample 8.83 8.00 8.28 Dilution factor of EVs 2.5 2.5 2.5 EVs (108 /ml) raw 22.08 20.01 20.69 Sybren L.N. Maas et al.
- 44. 29 3. For one of the files, filter the sample to display particles larger than the determined cutoff only. Set this sample as “calibra- tion” and enter the mode size of the calibration particles. 4. Couple the sample file and the newly create calibration file as outlined in Subheading 3.1 step 16. 5. Once successfully coupled, the unknown sample can now be displayed as a size distribution in nm based on the spiked cali- bration particles (Fig. 4, right). This graph will display two populations, one for the EVs and one for the reference particles. 4 Notes 1. For EV characterization different polystyrene beads are used: CPC100, CPC200, and CPC400, with mode diameters of 115, 203, and 335 nm, respectively (these numbers may vary based on the batch used). 2. Different sizes of nanopores are used: the NP100 nanopore (optimal size range 70–200 nm), NP150 (80–300 nm), and NP200 (100–400 nm). Due to heterogeneity of EV samples, the NP150 and NP200 are most often used for characteriza- tion of EVs. 3. To minimize background particle detection, we use filter-tip pipette tips. 4. To homogenize the calibration particles a basic tabletop soni- cator can be used. 5. To completely remove any residual liquids between measure- ments, lint-free tissue can be used. To minimize contamina- tion of background particles, lint-free tissue is preferred over regular tissues. 6. For almost all data analyses the Izon Control Suite can be used. However, all data-points can be exported for analysis in other software packages. For EV quantification using the spik- ing method, a spreadsheet software program is required. 7. Electronic devices used in close proximity of the instrument can significantly interfere with the detection signal. This inter- ference is observed as identical, quickly repeating short pulses. We have most often observed this interference caused by mobile phones. 8. This is done to decrease the risk of air-bubble formation in the lower fluid cell. Air bubbles can be a major source of instable baseline current. 9. The baseline current depends on the applied buffer, stretch and voltage. The current should be stable and the root mean square tRPS for EV Characterization
- 45. 30 (RMS) noise should be 10 pA. If these conditions are not met, air bubbles or (partial) nanopore blocking may be causative. To solve this, resuspend the sample in the upper fluid cell and check if the baseline becomes stable. If not, remove both the sample and the PBS in the lower fluid cell. If (after reapplication of PBS) no stable baseline current is established, the nanopore may be (partially) blocked. Tap the shielding cap (using the sup- plied plunger) to vibrate the nanopore and to disrupt particles. Clogging may also be solved by induction of a brief pressure by pushing down and pulling out of the plunger. Alternatively, the shielding cap can be put in place whilst pressing on the nano- pore, which will vibrate the nanopore. Also, the nanopore can be maximally stretched (i.e., 47 mm), combined with applying maximal external pressure. If still unsuccessful, remove the nanopore and rinse heavily using deionized water. Re-place the nanopore on the instrument. 10. Each nanopore has a target concentration. For the NP100 and NP150 nanopores the target concentration is 10E10 per ml and for the NP200 the target concentration is 10E9 per ml. 11. The blockade height caused by a particle moving through the nanopore is based on the stretch, the applied voltage and the buffer used. If the nanopore opening is reduced (less stretch applied) the relative blockade by the particle will increase. This also implies that smaller particles surpass the detection thresh- old. Larger particles, on the other hand, will block the nano- pore more frequently. By increasing the voltage applied, the flow of ions will increase and so will the (relative) blockade caused by particles moving through the nanopore. However, an increased voltage can also result in increased RMS noise. The flow of ions, and thus a higher baseline current, can also be established by using a buffer with increased salt concentra- tion. However, this may influence the EV characteristics, for instance caused by changes in osmosis. 12. For accurate detection of particles a mode blockade of at least 0.1 nA is required. However, the mode blockade set for the cali- bration particles will also determine the range of EVs detectable by the instrument. For instance, a mode blockade of 0.1 nA for 203 nm calibration beads indicates that the instrument will only be able to detect particles that are slightly smaller than 203 nm. Reducing the stretch or increasing the voltage (see Note 11) could be needed to decrease the lower detection limit. 13. External pressure needs to be applied to counteract the influ- ence of electrokinetic forces. These electrokinetic forces are not negligible when using small pore sizes (NP100-NP200) [14], which is often the case upon EV quantification. Since EVs display a modest zeta potential (i.e., the potential Sybren L.N. Maas et al.
- 46. 31 difference between the dispersion medium and the stationary layer of fluid attached to the particle) [15, 16] the influence of the electrokinetic forces is low and can be completely abol- ished by applying 0.8 kPa external pressure [13]. 14. The particle rate recorded (particles per minute) will depend on the concentration of the particles, the applied pressure and applied stretch (the rate will decrease by decreasing the stretch). Since at least 500 particles should be recorded, a particle rate of 100 per minute is advised but not required. In our experience particle rates 2000 per minute will be less reliable. 15. Multi-pressure measurement is advisable when measuring EVs with increased surface charge (e.g., as a result of coupling highly charged ligands to the surface). In such cases, difference in sur- face charge between EVs and polystyrene calibration beads will result in inaccurate concentration estimations as one of the par- ticle sets is more likely to move through the nanopore than the other [14]. Measurement of the calibration bead sample and EV sample at multiple pressures provides additional data that is used to accurately calculate the concentration of EVs. 16. Residual PBS in the upper fluid cell can dilute the measurement sample. To prevent this, remove the upper fluid cell and gently wipe lint-free tissue in the bottom-opening of the cell. 17. To accurately compare a calibration sample with an EV sam- ple, the baseline current should not differ more than 3%. If unable to reach a comparable baseline current, apply the strategy outlined in Note 9. Alternatively, dilution of the sam- ple in PBS could make the EV sample more comparable to the calibration sample. 18. Dilution in PBS may facilitate EV counting by the qNano instrument. However, to guarantee appropriate counting of EVs, try to keep the particle rate above 70–100 particles per minute (see Note 14). 19. Although not strictly necessary, an EV-to-bead ratio of approx- imately 1 will make the measurements most reliable. If EVs or beads outnumber their counterparts the calculation of con- centrations will be more prone to variation. 20. To distinguish EVs from polystyrene beads, both populations should be identifiable based on blockade sizes. For EV quantifi- cations in biological samples we tend to use a NP200 nanopore in combination with CPC400 (mode 335 nm) polystyrene beads or an NP150 nanopore in combination with 203 nm beads. To maximize the population of EVs detected, try to obtain settings where the polystyrene beads induce blockades of at least 0.5 nA. By increasing the blockade height caused by the polystyrene particles, the detection limit for the EVs will decrease (see Note 12). tRPS for EV Characterization
- 47. 32 21. Example sample preparation: (a) 40 μl cell culture supernatant (after 5 min 300×g centrifugation to remove cells). (b) 40 μl PBS. (c) 20 μl 1:200 diluted 203 nm polystyrene beads (stock 1e12 ml−1 ). 22. A beads-only sample is used to quantify background particles and to identify the population of polystyrene beads. For this sample “EV free cell culture medium” should be used that has received the same treatments as the samples of interest, but lacks EVs. 23. To spread variation in nanopore conditions, each set of sam- ples should be measured once before recording duplicates and triplicates. Prepare fresh samples (i.e., addition of PBS and beads) directly before each measurement. 24. Setting the cutoff remains arbitrary. Make sure each sample has the same bin-size setting (ViewSettings panel, accessible by clicking the popup button in the View panel). We choose to set the cutoff at 0.48 nA (Fig. 4, left). All particles smaller than the cutoff are determined EVs. 25. Since the EVs are diluted (upon mixing with calibration beads and addition of PBS) the obtained concentration should be corrected for this. For the example setup outlined in note 21, the EVs are diluted 2.5 times. 26. A correction can be introduced when the detection of EVs and polystyrene beads overlaps. Measure the EV sample with- out polystyrene beads and determine the “Bead-to-EV ratio” based on the cutoff determined in Subheading 3.2 step 8 (here the term “bead” refers to the fraction of EVs that are detected within the spiked-bead-detection range). Usually this ratio is insignificant, but if not add this Bead-to-EV ratio to the EV-to-bead ratio as determined in Subheading 3.2 step 13. This new ratio should be used for the remaining steps in the protocol. Acknowledgment We thank J. Berenguer (VUmc, Amsterdam, The Netherlands) for providing us with the glioblastoma cell culture supernatant. This work has been financially supported, in part, by the Dutch Hersenstichting (foundation concerned with diseases of the brain), the Schumacher Kramer Stichting (Foundation), and the TP Bohnenn foundation. Sybren L.N. Maas et al.
- 48. 33 References 1. Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3:22. doi:10.1002/0471143030.cb0322s30 2. Jorgensen M, Baek R, Pedersen S, Sondergaard EK, Kristensen SR, Varming K (2013) Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping. J Extracell Vesicles 2:PMID: 24009888. doi:10.3402/jev. v2i0.20920 3. de Vrij J, Maas SL, van Nispen M, Sena-Esteves M, Limpens RW, Koster AJ, Leenstra S, Lamfers ML, Broekman ML (2013) Quantification of nanosized extracellular membrane vesicles with scanning ion occlusion sensing. Nanomedicine 8(9):1443–1458. doi:10.2217/nnm.12.173 4. Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sargent IL (2013) Extracellular vesicle sizing and enumeration by nanoparticle tracking analy- sis. J Extracell Vesicles 2:PMCID: PMC3760643. doi:10.3402/jev.v2i0.19671 5. Nolte-'t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Quantitative and qualitative flow cyto- metric analysis of nanosized cell-derived membrane vesicles. Nanomedicine 8(5):712– 720. doi:10.1016/j.nano.2011.09.006 6. Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL (2011) Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7(6):780–788. doi:10.1016/j. nano.2011.04.003 7. van der Vlist EJ, Nolte-'t Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH (2012) Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantita- tive and qualitative analysis by high-resolution flow cytometry. Nat Protoc 7(7):1311–1326. doi:10.1038/nprot.2012.065 8. Maas SLN, De Vrij J, Broekman MLD (2014) Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing. J Vis Exp 92:e51623 9. Roberts GS, Kozak D, Anderson W, Broom MF, VogelR,TrauM(2010)Tunablenano/micropores for particle detection and discrimination: scanning ion occlusion spectroscopy. Small 6(23):2653– 2658. doi:10.1002/smll.201001129 10. Kozak D, Anderson W, Vogel R, Chen S, Antaw F, Trau M (2012) Simultaneous size and zeta-potential measurements of individual nanoparticles in dispersion using size-tunable pore sensors. ACS Nano 6(8):6990–6997. doi:10.1021/nn3020322 11. Vogel R, Willmott G, Kozak D, Roberts GS, Anderson W, Groenewegen L, Glossop B, Barnett A, Turner A, Trau M (2011) Quantitative sizing of nano/microparticles with a tunable elasto- meric pore sensor. Anal Chem 83(9):3499–3506. doi:10.1021/ac200195n 12. Willmott GR, Vogel R, Yu SS, Groenewegen LG, Roberts GS, Kozak D, Anderson W, Trau M (2010) Use of tunable nanopore block- ade rates to investigate colloidal dispersions. J Phys Condens Matter 22(45):454116. doi:10.1088/0953-8984/22/45/454116 13. Yang L, Broom MF, Tucker IG (2012) Characterization of a nanoparticulate drug deliv- ery system using scanning ion occlusion sensing. Pharm Res 29(9):2578–2586. doi:10.1007/ s11095-012-0788-3 14. Kozak D, Anderson W, Trau M (2012) Tuning particle velocity and measurement sensitivity by changing pore sensor dimensions. Chem Lett 41(10):1134–1136. doi:10.1246/cl.2012.1134 15. Marimpietri D, Petretto A, Raffaghello L, Pezzolo A, Gagliani C, Tacchetti C, Mauri P, Melioli G, Pistoia V (2013) Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression. PLoS One 8(9):e75054. doi:10.1371/journal.pone.0075054 16. Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M, Giebel B (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87(1):146–150. doi:10.1016/j. colsurfb.2011.05.013 tRPS for EV Characterization
- 49. 35 Chapter 3 Immuno-characterization of Exosomes Using Nanoparticle Tracking Analysis Kym McNicholas and Michael Z. Michael Abstract Due to their size, small extracellular vesicles such as exosomes have been difficult to identify and to quan- tify. As the roles that exosomes play in intercellular signalling become clearer, so does their potential utility as both diagnostic biomarkers for disease and as therapeutic vectors. Accurate assessment of exosomes, both their number and their cargo, is important for continued advancement in the field of vesicle research. To that end, several technologies, including nanoparticle tracking analysis, have been developed to define the physical characteristics of vesicle preparations and determine their concentration. This chapter describes a method for identifying the size and concentration of a subpopulation of vesicles in biological samples, using nanoparticle tracking analysis. Characterization of distinct exosomes is enabled by specific marker antibodies, coupled to fluorescent quantum dots. Key words Quantum dots, Qdots® , NanoSight, CD63, Exosomes, Microvesicles 1 Introduction Exosomes are extracellular vesicles, released from a wide variety of cell types, and are being increasingly recognized as mediators of intercellular signaling and potential biomarkers of human disease [1]. They comprise a specific subset of cell-derived vesicles, formed through the endosomal pathway [2]. The precise definition of an exosome is still open to some debate, even regarding their size. Early exosomal research was based on electron micrographs show- ing cup-shaped vesicles up to 100 nm in diameter, but more recent work has widened this range from 50-180 nm [3–5]. Regardless, it is their small size that poses unique challenges for their study. Nanoparticle tracking analysis (NTA) has been recently devised as a means of both quantifying and determining the size of particles between 30 nm and 1 μm in diameter. This analysis is reliant on a NanoSight (NanoSight Limited, Malvern Instruments, Amesbury, Andrew F. Hill (ed.), Exosomes and Microvesicles: Methods and Protocols, Methods in Molecular Biology, vol. 1545, DOI 10.1007/978-1-4939-6728-5_3, © Springer Science+Business Media LLC 2017
- 50. Other documents randomly have different content
- 51. [1] A hill near Paris famous for asses. Another time, while we were in the field, commanded by the great Sultan Erguebzed in person, and I, harassed by a forced march, was taking a nap in my tent, I thought I had the conclusion of an important affair to sollicite in the divan: I went to appear before the council of regency: but you may judge how much I had reason to be surprized. I found the hall full of racks, troughs, mangers, and coops for fowls; in the great Seneschal's easy chair I saw but an ox chewing the cud; in the Seraskier's place, a Barbary sheep; on the Testesdar's bench, an eagle with a hooked bill and long talons; instead of the Kiaja and Kadilesker, two large owls cloathed in fur; and for Visirs, geese with peacocks tails. I presented my petition, and instantly heard a horrible racket, which awaked me. Is that a dream of very difficult interpretation? said Mangogul, you had at that time some affair in the divan, and before you went thither, you took a walk to the Menagerie: but Signor Bloculocus, you tell me nothing concerning my dog's head. Prince, answer'd Bloculocus, 'tis a hundred to one, that madam wore, or you had observed some other lady wear a sable tippet; and that the first Dutch dog, which you saw, struck your imagination. There you have ten times more connections than is requisite to employ your mind during your sleep: the resemblance of colour made you substitute hair for a tippet, and in an instant you planted an ugly dog's head in the place of a very beautiful woman's head. Your notions to me appear just, replied Mangogul: why do you not publish them? they may contribute to the progress of divination by dreams, an important science, which was much cultivated two thousand years ago; and has since been too much neglected. Another advantage of your system is, that it would not fail throwing light on several works, both ancient and modern, which are but a string of dreams; such as Plato's treatise of idea's, the fragments of Hermes Trismegistus, the literary paradoxes of father Harduin, the Newton, the optic of colours, and the universal mathematicks of a certain Bramin. For example, would you not inform us, Mr. Conjurer,
- 52. what Orcotomus had seen in the daytime, when he dream'd his Hypothesis; what father C—— had dreamt, when he set about constructing his organ of colours; and what was Cleobulus's dream, when he composed his tragedy? With a little meditation, Sir, answered Bloculocus, I might compass all that: but I reserve these nice phænomena for the time, when I shall put out my translation of Philoxenus, for which I beseech your highness to grant me the privilege. With all my heart, says Mangogul: but who is this same Philoxenus?——Prince, replies Bloculocus, he is a Greek author, who was very knowing in the subject of dreams.——Then you understand Greek?——Who I, Sir, not a syllable.——Have you not told me that you are translating Philoxenus, and that he wrote in Greek? Yes, Sir; but in order to translate a language, it is not necessary to understand it: because translations are made for those only, who understand it not. That is wonderful, says the Sultan; Signor Bloculocus, well then translate Greek without understanding it. I give you my word, that I will keep the secret, and it shall not make me honour you one jot the less. CHAP. XL. Twenty-third Trial of the Ring. Fannia. There still remain'd a good part of the day, when this conversation was closed: which determined Mangogul to make one trial of his ring, before he retired to his appartment; tho' it were purely to fall asleep on more chearful idea's than those which had hitherto employ'd him. He went directly to Fannia's house; but found her not.
- 53. He return'd thither after supper; she was still absent. Wherefore he put off his experiment to the next morning. Mangogul, says the African author, whose Journal we translate, was at Fannia's house by half an hour after nine this morning. She was but just put to bed. The Sultan drew near her pillow, view'd her for some time, and could not conceive how, with so few charms, she had run through so many adventures. Fannia is fair even to insipidity, tall, ungainly, with an indecent gait, no features, few Agrémens, and an air of intrepidity, intolerable any where but at court. As for wit, she is allowed to have just as much as gallantry can communicate: and a woman must be born very weak, if she has not acquired a stock of jargon after a score of intrigues; for Fannia was advanced thus far. At this time she was possessed by a man suited to her character. He gave himself little or no concern about her infidelities; tho' indeed he was not as well informed as the public, how far she carried them. He had taken Fannia by caprice, and kept her by habit; like a piece of furniture. They had spent the night at the ball, went to bed at nine, and fell asleep without ceremony. Alonzo's indifference would not have suited Fannia, were it not for her easy humour. Thus our couple were sleeping soundly back to back, when the Sultan turn'd his ring on Fannia's Toy. It instantly began to speak, its mistress to snoar, and Alonzo to awake. After yawning several times; this is not Alonzo, what's o'clock, who wants me? your business, said the Toy. I think I have not been long in bed, let me take another nap. The Toy was preparing to compose itself to rest accordingly; but that was not the Sultan's intention. What persecution, resumed the Toy. Once more who wants me, and for what? 'tis a misfortune to be born of illustrious ancestors: how unhappy is the condition of a titled Toy! if any thing could console me for the fatigues of my state, it would be the goodness of the nobleman, whose property I am. Oh! he is certainly the best man in the world in that regard. He has
- 54. never given us the least uneasiness: and in return we have made great use of the liberty he granted us. What would have become of me, great Brama, if I had fallen to the share of one of those insipid wretches, who are always upon the watch? what a fine life we should have led! Here the Toy added some words, which Mangogul understood not, and then with surprising rapidity fell to sketching out a crowd of heroic, comic, burlesque, and tragicomic adventures: and it was almost out of breath, when it continued in these terms. You see I have some memory. But I am like all others; I have retained but the smallest part of what I have been intrusted with. Be satisfied therefore with what I have related to you, I can recollect no more at present. 'Tis pretty well, said Mangogul within himself; but still he urged afresh. Lud, how teizing you are, resumed the Toy: As if one had nothing better to do than to prate. Come then, since it must be so, let us prate on: perhaps when I have told all, I shall be permitted to do something else. My Mistress Fannia, continued the Toy, thro' an inconceivable spirit of retirement, quitted the court, to shut her self up in her house at Banza. It was then the beginning of autumn, and every body was out of town. And if you ask me what she did there; Faith, I can't tell. But Fannia never did but one thing; and if she had been employ'd that way, I should have known it. Probably she was out of work: true, I now recollect, we spent a day and a half in perfect idleness, which threw us into a cruel fit of the vapors. I was heart-sick of this sort of life, when Amisadar was so good to relieve us from it.—-'Ah! you are there, my poor Amisadar, indeed you give me great pleasure. You come to me very opportunely.'——'And who knew that you were at Banza?' replied Amisadar.—'No body truly; and neither you nor any one else will ever imagine what brought me hither. Don't you guess at the cause?'——'No, really, I cannot comprehend it.'—'Not at all?'—'No, not at all.'—'Well then know, my dear, that I resolved to be
- 55. converted'—'You, to be converted?'——'Yes, I'——'Look on me a little: but you are as charming as ever, and I see nothing in that countenance that bespeaks conversion. This is all pleasantry'——'No, faith, I am serious. I am determined to renounce the world. I am tired of it'——'This is a whim, that will soon fly off. Let me die, if ever you run into devotion'——'I will, I tell you: there is no sincerity in man'——'Pray has Mazul fail'd you?'——'I have not seen him this age.'——'Then it must be Zumpholo?'——'Less still, I have ceased seeing him, I can't tell how, without thinking about it.'——'Ah! I have it, 'tis young Imola?'—'Good, who can fix such fribbles?'—'What is it then?'——'I can't tell, I am angry with the whole earth?'——'Ah! Madam, you are in the wrong; for this earth, at which you are angry, might furnish you wherewithal to repair your losses.'——'Then, Amisadar, you sincerely believe that there are still some good souls, who have escaped from the corruption of the age, and are capable of love?'——'How, love! Is it possible that you give into those pitiful notions? you expect to be loved, you?'——'And why not?'——'But reflect, madam, that a man who loves, pretends to be loved, and alone too. You have too much good sense, to enslave your self to the jealousies and caprices of a tender and faithful lover. Nothing so fatiguing as these folks. To see but them, to love but them, to dream of none but them, to have no wit, humour, or charms but for them; all this most certainly does not suit you. It would be pleasant to see you stive yourself up in, what is called, the noble passion, and give your self all the awkward airs of a little female cit.' 'Well, Amisadar, you seem to be in the right. I verily think it would ill become us to run into fawning love. Let us change then, since it must be so. Besides, I do not see, that those loving women, whom they set us as models, are happier than others'——'Who told you so, madam?'——'No body, but it is easily foreseen.'——'Trust not to such foresight? A loving woman constitutes her own, and her lover's happiness: but this part is not suited to all women.'——'Faith, my dear, it is suited to none: for all, who attempt it, are sufferers. What advantage is there in fixing to one?'——'A thousand, a woman, who fixes her affections, will preserve her reputation; will be sovereignly esteemed by the man she loves; and you cannot imagine, how much
- 56. love owes to esteem.'——'I do not comprehend your meaning, you make a jumble of reputation, love, esteem, and I can't tell what besides. Would you be understood, that inconstancy must dishonour a woman? How, I take a man, and find he does not answer my expectations: I take another, and am still disappointed: I change him for a third, who does not turn out a jot better: and because I have had the misfortune to make a score of wrong choices, instead of pitying me, you would'——'I would, madam, advise a woman who has been deceived in her first choice, not to make a second; for fear of being deceived again, and running from one error into another.'——'Good God, what strange morality! I fancy, my dear, that you preached me a quite different sort just now. Might one be informed what sort of woman would hit your taste?'——'Most willingly, madam but 'tis late, and the discourse would run into too great a length.'——'So much the better: I am alone, and you will be company for me. Thus the affair is settled, is it not? Seat yourself on this couch, and go on: I shall hear you more at ease.' Amisadar obey'd, and sate down by Fannia. 'That mantelet of yours, madam,' says he, leaning towards her, and uncovering her bosom, 'wraps you up strangely.'—'You say right.'——'Why then do you hide such beautiful things?' added he, kissing them.——'Come, ha' done. Do you know that you are mad? You are become intolerably impudent. Mr. Moralist, resume the conversation which you began.' 'Well then,' said Amisadar, 'I would be glad to find in my mistress a good figure, good sense, good sentiments, and decency above all. I would have her approve my attendance; not deceive me by looks; make me thoroughly sensible, once at least, that I am agreeable to her; and even inform me how I may become still more so; not conceal from me the progress I make in her heart; give ear to none but me, have no eyes but for me; neither think, nor even dream, but of me; love but me; busy herself about nothing but me; do nothing but what may tend to convince me of all this: and at length yielding herself up to my transports, let me plainly perceive that I owe every thing to my love and to hers. Oh, what a triumph, madam! And how happy is the man who possesses such a woman!'——'Alas, my poor
- 57. Amisadar, you are certainly out of your senses. You have drawn the portrait of woman who does not exist.'——'Pardon me, madam, there are some in being. I own that they are rare; but yet I have had the good fortune to light of one. Alas! If death had not snatch'd her from me, for 'tis death alone that ever robs one of such women, perhaps I should be in her arms at present'——'But how then did you behave with her?'—'I loved to distraction, and miss'd no opportunity of giving her proofs of my passion. I had the sweet satisfaction of seeing that they were well received. I was scrupulously faithful to her, and she to me. The only disputes between us were, whose love was strongest; and in these little debates it was, that we laid our hearts open. We were never so fond as after this scrutiny of our souls. Our caresses always became more tender and vigorous after our explanations. Oh! what love and truth were then in our looks! I read in her eyes, and she in mine, that we burned with equal and mutual ardor.'——'And whither did all this lead ye?'——'To pleasures unknown to all mortals less amorous and sincere than us.'——'You enjoyed?'——'Yes I enjoyed, but a good on which I set an infinite value. If esteem does not intoxicate, at least it hightens the intoxication considerably. We unbosom'd ourselves without reserve, and you can't imagine how much it strengthened our passion. The more I examined, the more perfections I discovered, and the greater were my transports. I spent half my time at her feet, and I regretted the loss of the rest. I made her happiness, and she filled up the measure of mine. I always saw her with pleasure, and always quitted her with pain. Thus we lived together: and now, madam, you may judge if loving women are so much to be pitied'——'No they are not, if what you tell me be true; but I can scarcely believe it. There is no such love as you describe. Nay, I imagine, that such a passion as you have felt, must make a man purchase the pleasures it affords at the expence of great uneasinesses.'——'I had some, madam, but I was fond of them. I felt some twitches of jealousy. The least alteration which I remarked in her countenance, spread the alarm all over my soul.'——'What extravagance! Upon mature consideration, I conclude that it is better to love in the present fashionable way; to take a lover at one's ease,
- 58. keep to him while he amuses, quit him when he becomes tiresome, or that our fancy speaks for another. Inconstancy affords a variety of pleasures unknown to you languishing folks.'——'I grant that that method may be proper enough for little kept mistresses and common women; but does not suit with a man of tenderness and delicacy. At most it may amuse him, when his heart is disengaged, and he is willing to make comparisons. In a word, a woman of gallantry is by no means of my taste.'——'You are in the right, my dear Amisadar, you have a ravishing way of thinking. But do you love any thing at present?'——'Nothing, madam, but yourself; and I dare not tell you so neither.'——'Ah! my dear, dare on: you may continue,' replied Fannia, gazing on him stedfastly. Amisadar understood this reply thoroughly well, moved forward on the couch, fell to playing with a ribbon, which hung down on Fannia's breast, and he was not interrupted. His hand, meeting with no obstacle, slipt down lower. She continued to fire him with glances, which he did not misinterpret. For my part, says the Toy, I found, he was a sensible man. He took a kiss on that neck, on which he had bestowed so many encomiums. He was desired to stop, but in such a tone as plainly shewed that she would take it ill, if he obeyed; and accordingly he did not. He kissed her hands, returned to her neck, passed to her mouth: nothing resisted him. Insensibly Fannia's leg was on Amisadar's thighs. He put his hand on it: it was soft, and Amisadar did not fail to remark it. His elogy was heard with an air of distraction. By favor of this inattention, Amisadar's hand advanced, and with rapidity reached her knees. The absence of mind still continued; and Amisadar was preparing for the charge, when Fannia came to herself. She accused the little philosopher of want of respect; but he became so absent in his turn, that he did not hear one word, or at least made no other answer to the reproaches she threw on him, but by compleating his happiness. What a charming man he appear'd to me! Of the multitude of those, who preceded and followed him, not one was ever so much to my taste. I cannot mention him without panting. Pray suffer me
- 59. to recover breath! I think I have spoken a pretty sufficient time, considering it is my first speech. Alonzo did not lose one single Word of Fannia's Toy; and he was no less impatient than Mangogul to hear the remaining part of the adventure: but neither of them had time to be out of patience, when the tale-telling Toy resumed in these words. All that I can comprehend after serious consideration, is, that in some few days Amisadar went to the country, that he was asked the reason of his stay in town, and that he related his adventure with my mistress. For somebody of Amisadar's and her acquaintance, passing by our door, enquired either by chance or design, if madam was at home, sent in his name, and went up.——'Ah! madam, who could imagine you were in Banza? and how long are you here?'——'An age, my dear, this fortnight, that I have renounced society.' 'May I presume to ask, madam, upon what account?'——'Alas! because I was tired of it. Women are become such strange libertines, that there is no bearing them. One must either do as they do, or pass for a silly creature; and sincerely, I think both extremes should be avoided.'——'Indeed, madam, you are become quite edifying. Pray, is it the conversation of the Bramin Brelibibi, that has wrought your conversion?'——'No, 'tis a squall of philosophy, 'tis a quint of devotion. It seized me suddenly; and it is not poor Amisadar's fault that I am not at present practising the highest austerity.'——'Then madam has seen him lately?'——'Yes, once or twice.'——'And you have seen no body else.'——'No, truly. He is the only thinking, reasoning, active being, that has entered my doors during the eternity of my retreat.'——'That is singular'——'And what singularity is there in it?'——'Nothing but an adventure which he had the other day with a lady of Banza, alone like you, devout like you, retired from the world like you. But I must tell you the story: perhaps it will amuse you.'——'Without doubt,' replied Fannia: and immediately Amisadar's friend set about relating his adventure word for word, as I have done, says the Toy: and when he was advanced as far as I am now.——'Well, madam,' said he, 'what do you think? Is not Amisadar a lucky man?'——'But,' answered Fannia,
- 60. 'Amisadar is a lier perhaps: do you imagine that there are women so daring as to abandon themselves without shame?'——'But consider, madam,' replied Maruspha, 'that Amisadar has named no body, and it is very improbable that he has imposed'——'I begin to see thro' the affair,' says Fannia: 'Amisadar has wit, and is a handsome man, he has, to be sure, infused some notions of sensual pleasure sure into this poor recluse, which have mastered her. Yes, this must be it: this sort of folks are dangerous to hear, and Amisadar is matchless in that way.'——'How, madam,' interrupted Marsupha, 'is Amisadar the only man that has the art of perswading, and will you not do justice to others, who deserve, as much as he, a share in your esteem?—- 'Pray, whom do you mean?'——'Myself, madam, who think you a charming woman, and'——'I fancy you joke. Look at me then, Marsupha. I have neither paint nor patches. My night-cap does not become me. I make a frightful figure.'——'You are mistaken, madam: that undress sets you off surprisingly. It gives you so winning and kind an air!'—— To these gallantries Marsupha added others. I insensibly joined in the conversation; and when Marsupha had finished with me, he resumed with my mistress. 'Seriously, Amisadar has attempted your conversion; he has an admirable hand at conversions. Could you give me a sample of his morals? I would lay a wager they are much the same with mine.'——'We have thoroughly handled some points of gallantry. We have analysed the difference between an affectionate woman and a woman of gallantry. He is for the affectionate women'——'And you too without doubt?'——'Not at all, my dear. I took great pains to demonstrate to him, that we were all alike, and that we acted upon the same principles: but he is not of this opinion. He establishes an infinity of distinctions, which, I think, exist nowhere but in his imagination. He has formed to himself, I can't tell what ideal creature, a chimæra of a woman, a non-entity in a coif.'——'Madam,' answered Marsupha, 'I know Amisadar. He is a lad of good sense, and has been very conversant with the sex. If he has told you that there were such'——'Oh! whether there are such or not,' interrupted Fannia, 'I could never conform to their
- 61. customs.'——'I believe it,' said Marsupha: 'and accordingly you have chosen another sort of conduct more suitable to your birth and merit. Those silly creatures are to be abandoned to philosophers: they would never be look'd on at court.—— Here Fannia's Toy stopt short. One of the principal perfections of these orators was to break off their discourse à propos. They talk'd as if they had never done any thing else: whence some authors have inferr'd, that they were pure machines. In this place the African author specifies all the metaphysical arguments of the Cartesians against the soul of brutes, which he applies with all possible sagacity to the prating of Toys. In a word, his opinion is, that Toys speak as birds sing; that is to say, so perfectly without having been taught, that, to be sure, they are prompted by some superior intelligence. But you ask me how he disposes of his prince. He sends him to dine with the favorite: at least 'tis there we shall find him in the following chapter. CHAP. XLI. The history of Selim's travels. Mangogul, whose thoughts ran solely on diversifying his pleasures, and multiplying the trials of his ring; after having interrogated the most interesting Toys of the court, had the curiosity to hear some of the city Toys. But as he had no advantageous opinion of what his should learn from them, he would willingly consult them at his ease, and save himself the trouble of going to find them out. How to bring them to him, was what embarassed him. You are in great pain for a trifle, says Mirzoza. Sir, you have only to give a ball, and I promise you this very night a greater number of those speech-makers than you will covet to hear.
- 62. My heart's joy, you say right, replied Mangogul; and your contrivance is the better still, because we shall certainly have none but those whom we have occasion for. In a moment an order was dispatch'd to the Kislar-Agasi, and the master of the revels, to prepare the ball, and to distribute no more than four thousand tickets. They were probably better judges in that country than elsewhere, of the room that six thousand persons would take up. To amuse themselves till the hour of the ball, Selim, Mangogul, and the favorite set about telling news. Does madam know, says Selim to the favorite, that poor Codindo is dead? This is the first word I heard of it, but what did he the of? says the favorite. Alas, madam, answered Selim, he fell a victim to attraction. He filled his head with this system in his youth, and it turn'd his brain in his old days. How so? says the favorite. He had found, continued Selim, by the methods of Halley and Circino, two celebrated astronomers of Monoémugi, that a certain comet, which made so much noise towards the end of Kanaglou's reign, was to return the day before yesterday; and fearing lest it might double its steps, and he should not have the happiness of being the first to see it; he resolved to spend the night in his observatory, and yesterday morning at nine o'clock he had still his eye clung to the telescope. His son apprehending the consequences of so long a sitting, went to him at eight, pull'd him by the sleeve, and called him several times: Father, Father. Not a word of answer. Father, Father, repeated the young Codindo. 'It is just going to appear,' replied Codindo: 'it will appear; zounds! I shall see it.' 'But you do not consider, dear father, that there is a dismal fog'——'I must see it, I will see it, I tell thee.' The young man, convinced by these answers, that the fog had got into his father's head, called out for help. The family ran to him, and sent for Farfadi; and I was with him (for he is my physician) when Codindo's servant came.——'Quick, quick, Sir, make haste, old Codindo, my master'——'Well, what is the matter, Champagne? What
- 63. has befallen your master?'——'Sir, he is run mad.'——'Thy master is run mad'——'Oh! yes, Sir. He cries out that he must see beasts, that he will see beasts; that they will come. The apothecary is with him already, and they wait for you. Come quickly.'——'Maniacal,' says Farfadi, putting on his gown, and hunting for his square-cap; 'Maniacal, a terrible maniacal fit.' Then turning to the servant, he ask'd: 'Does not thy master see butterflies? Does he not pick the ends of his coverlid?'——'Oh! no, Sir,' replied Champagne. 'The poor man is on the top of his observatory, where his wife, daughters and son have much ado to hold him. Come quickly, you will find your square-cap to-morrow.' Codindo's disease seemed to me to be of an odd kind: I took Farfadi in my coach, and we drove to the observatory. At the bottom of the stairs we heard Codindo crying out in a furious tone: 'I must see the comet, I will see it: withdraw ye rascals and jades.' In all probability his family, finding that they could not prevail on him to go down to his bed-chamber, had ordered his bed up to him: for we found him lying in bed at the top of his observatory. An apothecary of the neighbourhood, and the Bramin of the parish had been called before we arrived. The latter was trumpeting into his ear: 'Brother, dear brother, your salvation is at stake: you cannot with a safe conscience expect a comet at this hour of the day: you damn yourself.'——'That is my business,' said Codindo. 'What answer will you give to Brama, before whom you are going to appear?' replied the Bramin.——'Mr. Rector,' says Codindo, without stirring his eye from the telescope, 'my answer shall be, that it is your trade to exhort me for my money, and the apothecary's there, to extol his warm water to me; that the physician does his duty of feeling my pulse, and learning nothing from it; and I my own, of waiting for the comet.'——In vain did they teize him, they drew nothing more from him: he continued to observe with heroic courage; and he died on the leads, his left hand on his eye of that side, his right laid on the tube of the telescope, and his right eye applied close to the eye- glass; between his son, who cried that he made a false calculation; his apothecary, who proposed him a clyster, his physician, who with
- 64. a toss of his head pronounced, that there was nothing more to be done; and his priest, who said to him: 'brother, make an act of contrition, and recommend yourself to Brama.——' That is, says Mangogul, what they call dying in the bed of honour. Let us leave poor Codindo, added the favorite, to rest in peace, and pass to some more agreeable subject. Then addressing herself to Selim, my lord, says she, as you are so gallant at this time of life, have so much wit, talents, and so good a mien, and lived in a court devoted to pleasures; it is no wonder if the Toys have formerly celebrated your fame. But yet I suspect that they have not told all they knew of you. I do not require this supplement: you may have good reasons for refusing it. But after all the adventures, with which this gentry have honoured you, you ought to know womankind: and this is one of those things of no consequence, which you may safely own. This compliment, madam, replied Selim, would have flattered my self-love at the age of twenty: but I have gained some experience, and one of my first reflections is, that the more one practises this business, the less knowledge he obtains. I, to know women! that I have studied them much, may be allowed. Well, what do you think of them? said the favorite. Madam, answered Selim, whatsoever their Toys might have published concerning them, I esteem the whole sex as most respectable. Indeed, my friend, says the Sultan, you deserve to be a Toy; you would have no occasion for a muzzle. Selim, added the Sultana, abandon the satyrical strain, and speak the truth. Madam, replied the courtier, I may possibly mix some disagreeable strokes with my narrative: do not impose the task on me of offending a sex, which has always used me well enough, and which I revere by——What, always veneration! I know nothing so caustic as those sweet- tongued folks, when they set on, intermitted Mirzoza; and imagining that it was through regard for her that Selim excused himself, Let not my presence restrain you, added she: we are contriving to amuse ourselves; and I promise upon my honour to
- 65. apply to myself all the obliging things you shall say of my sex, and to leave the rest to other women. Well, you have studied women much? Pray, give us an account of the course of your studies: it must have been very brilliant, if I may judge of it by what is known of the success: and it is reasonable to presume, that this will not be contradicted by what is unknown. The old courtier complied with her desire, and began thus. The Toys, I own, have talked a good deal of me: but they have not told all. Those who were capable of completing my history, either are no more, or are not in our climate: and those who have begun it, have but lightly touched the subject. I have hitherto inviolably kept the secret which I had promised them; although I was better made to speak than they: but since they have broke silence, I think they have dispensed me from the obligation of keeping it. Born with a fiery constitution, I loved almost as soon as I knew what a beautiful woman was. I had governants which I detested; but in return I was much pleased with my mother's waiting-women. They were for the most part young and pretty: they conversed, dressed, and undressed before me without ceremony; they have even enticed me to take liberties with them, and my temper naturally inclining to gallantry, turned every thing to advantage. With these elements of instruction, at five or six years of age I was put under the care of men; and God knows how forward I was in improving them, when the ancient authors were put into my hands, and my tutors explained certain passages, of which possibly they themselves did not penetrate into the sense. My father's pages taught me some pretty college tricks: and the perusal of Aloysia, which they lent me, gave me a vehement desire of becoming perfect. I was then fourteen years of age. I cast my eyes around, seeking among the women who frequented the house, one to whom I might make my addresses: but they all appeared equally proper to ease me of my irksome load of innocence. A commenced acquaintance, and still more the courage I felt to attack a person of my own age, and which failed me with
- 66. regard to others, determined my choice in favor of one of my cousins. Emilia was young, and so was I: I thought her pretty, and she liked me: she was not difficult, and I was enterprizing: I had a mind to learn, and she was not less curious to know. We frequently asked one another very frank and strong questions: and one day she deceived the vigilance of her governants, and we instructed each other. Ah! how great a master is nature! it soon set us in the high road of pleasure, and we abandoned ourselves to its impulse, without the least thought of the consequences: and this was not the way to prevent them. Emilia had indispositions, which she took the less pains to hide, as she did not suspect the cause. Her mother examined her, extorted a confession of our commerce, and my father was informed of it. He made me some reprimands blended with an air of satisfaction; and it was immediately resolved that I should travel. I set out with a governor, who was charged to watch my conduct attentively, but not to put me under any restraint: and five months after, the gazette informed me, that Emilia died of the small pox; and a letter from my father, that her tenderness for me had cost her her life. The first fruit of my love serves with distinction in the Sultan's army: I have always supported him by my credit, and to this day he knows me solely as his protector. We were at Tunis, when I received the news of his birth and his mother's death. Her fate touched me to the quick, and I believe I should have been inconsolable, had I not embarked in an intrigue with a sea-captain's wife, who did not afford me time to run into despair. The Tunetine was intrepid, and I was fool-hardy: for with the assistance of a rope-ladder, which she threw to me, I passed every night from my lodging on her terrass, and thence into a closet, where she put the finishing hand to my instructions; Emilia having only made a beginning. Her husband return'd from a cruize, just at the time, that my governor, who had received his instructions, urged me to cross over into Europe. I embarked on board a vessel bound for Lisbon, but not without several times taking leave of Elvira, from whom I received this diamond.
- 67. The vessel, in which we sailed, was laden with merchandise; but the most valuable commodity on board, to my taste, was the captain's wife. She was not quite twenty: and her husband was as jealous of her as a tyger, and not quite without cause. We all soon understood one another: Donna Velina perceived that I had a liking for her; I, that I was not indifferent to her; and her husband, that he incommoded us. The sailor resolved not to lose sight of us till we were landed at Lisbon. I read in the eyes of his dear wife, how much she fretted at her husband's assiduity: mine testified the same things to her, and the husband understood us wonderfully well. We spent two whole days in an inconceivable thirst of pleasure; which would certainly have kill'd us, had not heaven assisted us: but it always assists souls in pain. Just upon our passing the Streights of Gibraltar, a furious tempest arose. I would not fail, madam, to raise the winds about your ears, and make thunder rattle over your head; to set the heavens on fire with lightning; raise the billows up to the clouds, and describe the most horrid tempest which you have ever met with in any romance; were I not giving you a history. I shall only tell you, that the captain was compelled by the sailors cries to quit his room, and expose himself to one danger for fear of another. He went up on deck together with my governor, and I threw myself without hesitation into the arms of my fair Portuguese; quite forgetting that there was any such thing in nature as a sea, storms, or tempests; that we were on board a tottering vessel; and abandoning myself without reserve to the perfidious element. Our course was rapid, and you may well judge, madam, by the weather at that time, that I saw a great deal of land in a few hours. We put in at Cadiz, where I left a promise with the Signora to meet her at Lisbon, if my Mentor agreed to it, whose design was to go directly to Madrid. The Spanish women are more closely confined, and more amorous than ours. Love is managed in that country by a sort of ambassadresses, who have orders to catechize strangers, to make proposals to them, to conduct them forward and backward; and the ladies undertake the task of making them happy. I was not obliged
- 68. to go through this ceremony, thanks to the conjuncture. A great revolution had lately placed a prince of the blood royal of France on the throne of this kingdom: his arrival and coronation occasioned festivals at the court, where I then appeared. I was accosted at a masquerade; and a meeting was proposed me for the next day: I accepted the challenge, and went into a little house, where I found only one man mask'd, his nose wrapp'd in his cloak, who delivered me a letter, in which Donna Oropeza put off the party to the next day at the same hour. I returned, and was introduced into an appartment sumptuously furnish'd, and well illuminated with wax tapers. My goddess did not make me wait long. She enter'd just at my heels, and rush'd into my arms without speaking a word, or taking off her mask. Was she ugly? Was she handsome? was what I knew not. I only perceived on the couch, to which she drew me, that she was young, well-made, and loved pleasure. When she found herself satisfied with my panegyricks, she unmask'd, and shewed me the original of this picture, which you see in my snuff-box. Selim open'd, and at the same time presented the favorite with a gold box, of exquisite work, and richly adorn'd with jewels. The present is gallant, says Mangogul: what I esteem most in it, added the favorite, is the portrait. What eyes! What a mouth! what a neck! But is not all this hightened? So little, madam, replied Selim, that Oropeza would probably have fixed me at Madrid, if her husband, informed of our commerce, had not disturbed it by his threats. I loved Oropeza, but I loved life better still. Besides, my governor was not of opinion, that I should expose myself to be poniarded by the husband, for the sake of enjoying his wife some few months more. Wherefore I wrote to the fair Spanish Donna a very moving farewel letter, which I stole out of some romance of that country, and set out for France. The monarch, who then reigned in France, was the king of Spain's grandfather, and his court was justly esteemed the most magnificent, most polite, and most gallant in Europe. I appeared there as a phænomenon. 'A young lord of Congo,' says a beautiful marquise. 'That must be surely very diverting: those men are better
- 69. then ours. I think Congo is not far from Morocco.' Suppers were given, to which I was invited. Let my discourse have ever so little sense in it, it was found fine, admirable: people retracted, who had at first done me the honour to suspect that I had not common sense. 'He is a charming man,' says another court lady briskly: 'it would be murther to suffer so pretty a figure to return into a wretched country, where the women are narrowly watched by men who are no longer so. Is it true, sir? 'Tis said, that they have nothing. That is very unseemly in a man.'——'But,' adds another, 'we must keep this great boy here, (for he is well born) tho' he were only made a knight of Malta. I engage, if you will, to procure him an employment; and the dutchess Victoria, my old friend, will speak to the king in his favor, if it be requisite.' I soon had indubitable proofs of their good-will, and I put the marquise into a condition of pronouncing on the merit of the inhabitants of Morocco and Congo. I found that the employment, which the dutchess and her friend had promised me, was difficult to execute, and therefore gave it up. It was in this recess that I learned to form those noble passions of twenty-four hours. I circulated during six months in a vortex, where the beginning of an adventure did not wait for the end of another; because enjoyment was the only thing intended. Or if that was slow in coming, or as soon as it was obtained, we ran upon the scent of new pleasures. What do you tell me, Selim? interrupted the favorite. Decency is then unknown in those countries? Pardon me, madam, replied the old courtier. They have scarcely any other word in their mouths. But the French women are no more slaves to the thing than their neighbors. What neighbors? says Mirzoza. The English women, replied Selim, who are cold and scornful in appearance, but passionate, voluptuous, vindictive; less witty and more rational than the French women. These love the jargon of sentiment, those prefer the expression of pleasure. But at London as at Paris, people love, separate, rejoin to separate again. From the daughter of a lord bishop (these are a sort of Bramins who do not keep celibacy) I passed to a baronet's wife. While he was warmly supporting the interest of the nation in the
- 70. house of commons, against the attempts of the court; his wife and I had quite different debates in his house. But the session was closed, and madam was obliged to attend her knight to his manor. I then light upon a colonel's wife, whose regiment was quartered along the sea-coast: I afterwards belong'd to the lady mayoress. Ah, what a woman! I should never have seen Congo again, if the prudence of my governor, who saw me wasting away, had not redeemed me from this gally. He counterfeited letters from my family, which recalled me with all possible expedition, and we embarked for Holland: our design was to travel through Germany into Italy, where we expected frequent opportunities of vessels to carry us to Afric. We saw Holland only in riding post; and did not tarry much longer in Germany. All the women of rank there resemble important citadels, which must be besieged in form. They are to be reduced, but the approaches require so many measures, there are so many ifs and buts, when the articles of capitulation are to be settled, that those conquests soon tired me. I shall never forget the expression of a German lady of the first quality, on the subject of granting me what she had not refused to several others. 'Alas!' cried she mournfully, 'what would my father the great Alkizi say, if he knew that I abandon myself to such a low creature as a Congese.' 'He shall say nothing, madam,' replied I: 'so much grandeur affrights me, and I withdraw.' It was wisely done of me; for if my mediocrity had compromised with her highness, I might have repented it. Brama, who protects the wholesome climes, which we inhabit, inspired me without doubt in this critical moment. The Italian ladies, whom we frequented afterwards, are not mounted on so high a pin. It was with them that I learned the modes of pleasure. There is indeed much caprice and whim in those refinements; but you will pardon me, ladies, if I say, that sometimes there is no pleasing you without them. From Venice and Rome I brought some merry receipts before my time unknown in our barbarous country. But I restore all the glory of them to the Italian women, who communicated them to me.
- 71. I spent about four years in Europe, and returned through Egypt into this empire, modelled as you see, and stock'd with the rare secrets of Italy, which I soon divulged. Here, says the African author, Selim perceiving that the common place language, which he held to the favorite on his adventures in Europe, and on the characters of the women of the countries through which he passed, had plunged Mangogul into a deep sleep, was afraid of awaking him; and therefore drew near to the favorite, and continued in a lower voice. Madam, said he, were I not apprehensive that I have tired you by a narrative, which has perhaps been already too long; I would relate you the adventure, by which I commenced my operations on my arrival at Paris: I cannot think how it has escaped me. Tell it, my good friend, answered the favorite; I will double my attention, and make amends, as much as I am able, for the Sultan's inattention, who sleeps. At Madrid, continued Selim, we had taken recommendations for some lords of the court of France, and at our setting foot in Paris we found ourselves loaded with protestations of friendship. It was then the pleasant season of the year, and in the evenings my governor and I went to walk in the gardens of the Palais Royal. One day we were joined there by some Petits-Maitres, who shewed us the most celebrated beauties, and gave us their history, true, or false, not forgetting themselves on every occasion, as you may well imagine. The garden was already stock'd by a great number of women; but there arrived a considerable reinforcement about eight o'clock. By the quantity of their jewels, the magnificence of their dress, and the crowd of their attendants, I took them for dutchesses at least. I spoke my thoughts to one of the young lords of the company, whose answer was, that he found I was a connoisseur; and if I was inclined, I should have the pleasure of supping that very night with some of the most lovely of them. I accepted his offer, and in an instant he slipt a word into the ears of two or three of his friends, who dispersed themselves into different parts of the walks, and in
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