![6 Oheim
6. Marine Biological Association, Plymouth, The Microe-
lectrode Techniques for Cell Physiology, http://
www.mba.ac.uk/events.php
7. European Molecular Biology Organization, Practical
Courses, EMBO Course on Light Microscopy in Living
Cells, Heidelberg, http://www.mba.ac.uk/events.php
A more comprehensive list is found by linking to
http://www.olympusfluoview.com/resources/courses.html
1.5. Free Photonics
Journals
Another valuable source of information for beginning as well as
confirmed microscopists is freely available monthly journals. A
particularity of the photonics market, there are quite a few of
them. Although often in close (sometimes all-too-close) prox-
imity with advertiser and manufacturer opinion, these publica-
tions are a showcase of recent developments in optics, microscopy,
and biophotonics. They provide up-to-date information on new
equipment, notable technical achievements, and provide an excel-
lent overview of trade fairs and meetings, both to come (which
is good if you are to chose components for your microscope),
or in the form of brief synopses, reviewing recent trends. While
not replacing the academic literature, they certainly broaden your
horizon and keep you connected to the often rapidly evolving
technology at no extra cost.
1. Photonics Spectra
2. Europhotonics
3. Biophotonics International, all three from Photonics
Media, Laurin Publishing, http://www.photonics.com
4. BioTechniques, from Informa Healthcare, http://www.
biotechniques.com
5. Optics & Laser Europe, IOP Publishing, http://
www.optics.org
6. Photonik international (German) from AT-Fachverlag,
http://www.photonik.de
7. Photoniques, de la Société Française d’Optique,
http://www.photoniques.com
1.6. Keep Your Cells
Alive
When imaging live samples, the first question arising is how to
use the limited photon budget without compromising sample
viability. Careful controls should be made to develop a quan-
titative notion of how the used intensities affect the biological
process under study. The result is often surprising because pho-
todamage starts gradually before obvious signs occur. For exam-
ple, two studies investigating how ample was two-photon photo-
damage when imaging intracellular free calcium ([Ca2+
]i) (1, 2)
found that the slope and kinetics of neuronal calcium signals
were attenuated much earlier than electrophysiological signs of](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-19-320.jpg)
![10 Oheim
Also exists in a 20 × 20-mm nanobench version. Unfor-
tunately, the future of the even larger macrobench (150 ×
150 mm) is with a question mark.
9. AHF (http://ahf.de) has the missing part for the
microbench: a 45◦
holder for a 1-mm 25 × 63-mm (Zeiss
or Olympus) standard dichroic mirror.
10. Interestingly, Thorlabs offers a fair number of cage
system components that are largely compatible with
the Microbench, thus considerably enlarging the choice
of optical components, http://www.thorlabs.com/
navigation.cfm?Guide ID=2002.
2. Instrumenta-
tion
2.1. Understanding
the Building Blocks
of the Laboratory
Microscope
It is quite instructive to forget what you know about laboratory
microscopes for a moment. Modern fluorescence microscopes are
highly modular and can be thought of as a box with lots of arms
sticking in and out (Fig. 1.2). Excitation arms can be different
channels of epi-illumination, a laser injected through a side port
for total internal fluorescence or photoswitching, a spinning-disc
confocal attachment, or a pulsed UV-lamp for flash-photolysis.
For a given excitation channel i and fluorophore j, excita-
tion is fully described in terms of the source spectral emission
S(λ), transmission of the excitation filter EX(λ), and the reflectiv-
ity diachroic beamsplitter (1–BS(λ)), which, for a give excitation
channel, are multiplied along the excitation optical path:
exi (λ) = S(λ) · EXi (λ) · (1 − BS(λ)) [1]
combined with the sample molar extinction εj and absorbance
spectrum Ej(λ) and integrated over λ to give an excitation spectral
function:
ξi j =
dλ exi (λ) · E j (λ) · εj . [2]
On the emission site, one proceeds analogously for each
detection arm k by multiplying the fluorophore quantum yield
and emission spectrum, spectral transmission of the dichroic and
emission filters, and the detector spectral sensitivity:
emk(λ) = BS(λ) · EMk(λ) · D(λ). [3]
Upon integration,
ξ
jk =
dλφ · Fj (λ) · emk(λ) [4]](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-23-320.jpg)
![Imaging Instrumentation and Formats 11
Finally, the product of eqs. [2] and [1.4],
ξi jk ≡ ξi j ξ
jk [5]
measures the signal of one mole/l of fluorophore j viewed
through channel k upon excitation in channel i, and similarly for
all permutations ijk.
Optimizing the photon yield now consists in maximizing
the detection spectral function, while balancing the excitation
spectral function with that of other fluorophores present in the
sample.
This balancing can be achieved not only by choosing appro-
priate filters but also by considering the source and detector spec-
tra and the microscope intermediate optics and objective lens.
Their transmission can conveniently be combined with that of
the beamsplitter, thereby accounting for the double passage of
excitation and emission light through the microscope.
The following list provides a quick overview of the principal
choices available and briefly discusses their respective advantages
and disadvantages with respect to the scheme shown in Fig. 1.2.
D(λ)
S(λ)
EM(λ)
EX(λ)
BS(λ)
M excitation channel(s) N fluorophore(s) L detection channel(s)
S(λ) · EX1(λ) · (1–BS(λ)) E1(λ) · (ε1φ1) · F1(λ)
E2(λ) · (ε2φ2) · F2(λ)
S(λ) · EX2(λ) · (1–BS(λ)) BS(λ) · EM2(λ) · D(λ)
BS(λ) · EM1(λ) · D(λ)
…
…
…
a
b
Fig. 1.2. (a) Schematic respresentation of an upright epifluorescence microscope. S – source; EX – excitation filter; BS –
diachroic beamsplitter; EM – emission filter; D – detector. See main text for details. (b) Box plot of the different excitation
and emission channels along with their spectral throughput, obtained by multiplying instrument and fluorophore spectral
properties along the excitation and detection arms. See main text for details.](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-24-320.jpg)
![Imaging Instrumentation and Formats 13
λ(θ) = λ0
1 − (sinθ/neff)2, [6]
For angles θ increasing beyond 45◦
, neff is determined by
the dichroic of the order of 1.7–1.86 for most of the coatings,
but varies for p- or s-polarized light so that their average must
typically be considered for the collected fluorescence.
The microscope intermediate optical path (tube lens, scan lens,
epi-illuminator, projection lenses in the case of compound mag-
nifiers) and the objective are often not considered in detail, until
unanticipated losses occur. For example, both UV and near-IR
transmission become a problem, e.g., in fura-2 Ca2+
imaging,
flash photolysis, or photoactivation, as well as two- or three-
photon microscopy using far-IR excitation.
• UV transmission. With the increasing demand for highly
corrected objectives (and tube lenses), high refractive index
glasses and optical cement are often used chromatic correc-
tions that limit UV transmission. Also, with microelectron-
ics getting smaller and smaller, the formerly used UV-light
inspection techniques often fail because of resolution limita-
tions. Thus, dedicated UV-transmitting optics is getting rarer
and rarer.
• Another often overlooked aspect is limiting intermediate
apertures (filter cubes, lenses, irises) that reduce the col-
lected light fraction of scattered fluorescence photons in
two-photon microscopy. While scattered photons are usu-
ally rejected in fluorescence imaging (because they are not
assigned to the pixel of their origin and hence blur the image)
they constitute useful signal in 2PEF microscopy that seeks
to optimize the light collection. Thus, keeping the product
of d sin(θeff) constant is of crucial importance for not using
light. Here, d is the size of the imaged spot and θeff is the
half-angle of the effective numerical aperture.
Much of the same reasoning is true for choosing and optimiz-
ing detectors. Know your microscope is the rule toward getting
optimal results.
2.2. The Performance
Triangle
Irrespective of all these parameters, you can only distribute your
collected photons once. Thus, whenever you take an image of
a live cell, you take from the budget of photons that your sam-
ple emits before irreversibly undergoing photodamage and pho-
todestruction. If you invest them into higher spatial resolution,
higher temporal resolution or larger image contrast (signal-to-
noise ratio) is your choice. And it is often a difficult one.
Resolution vs. magnification. On most available microscopes,
resolution is diffraction-limited. Thus, the smallest distance that
two objects can be close by and still be detected as two is defined
by the numerical aperture of the objective, according to Abbe’s](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-26-320.jpg)
![Labels and Probes for Live Cell Imaging 19
signals from these dyes can be difficult to separate from tissue
autofluorescence. In contrast, today there is a vast and often con-
fusing array of fluorophore labels available to scientists. These flu-
orophores span the optical spectrum from the UV to the visible
and extend into the near infrared (6–12). Many of the currently
available amine reactive fluorophores are summarized in Fig. 2.1
and the structures of some representative labels are shown in Fig.
2.2. Several factors must be considered in choosing the appropri-
ate fluorophores for constructing effective imaging agents. These
include method of attachment to the targeting group, excitation
and emission wavelengths, brightness, hydrophilicity, and cost.
There are many current chemistries available for the coupling
of fluorescent labels to biomolecules and targeting groups (Fig.
2.3). The most frequently employed synthetic handle for biocon-
jugation is the succinimidyl ester, which forms stable amide bonds
after reaction with primary and secondary amines. The isothio-
cyanate group may also be used for coupling to amines, generat-
ing a thiourea linkage. In cases where reaction of a succinimidyl
ester or isothiocyanate derivatized fluorophore with an amine is
not feasible, additional coupling groups are available. Iodoac-
etamide, maleimide, and dithiol-modified fluorophores are use-
ful for covalent conjugation to thiols. Hydrazine and hydrazide
modified dyes can be used for coupling to aldehydes and ketones,
forming relatively stable hydrazone linkages. More recently, the
development of bioorthogonal coupling schemes has attracted
significant interest for preparation of fluorescent probes.
Bioorthogonal couplings rely on use of reaction partners that
display little or no reactivity with common biological materials.
Two examples of these reactions are the Staudinger ligation (13)
and the “click” reaction (14). The Staudinger ligation involves
coupling of a methyl ester electrophilic trap with an azide to
generate an amide linkage and one equivalent of N2. This reac-
tion is mediated by oxidation of an adjacent phosphine. The click
reaction is a copper(I) catalyzed [3+2] cycloaddition between an
azide and an alkyne that results in the formation of a stable tri-
azole product (14). This reaction has excellent potential for use
in design of targeted fluorescent probes. However, there are only
a few azide or alkyne modified dyes currently available for this
reaction, most of which emit in the visible region. The poten-
tial utility of the click reaction in biology suggests that in the
coming years the selection of azide and alkyne modified dyes is
likely to expand greatly. For example, recent efforts have yielded
new, efficient synthetic routes to far-red/near infrared emitting
cyanine dyes modified with either azide or alkyne groups, one
example of which, CyAM-5 alkyne, is shown in Fig. 2.2 (15).
Although highly selective, cytotoxic copper(I) is necessary for
the traditional click coupling, and therefore direct use of this
reaction in living biological systems has not been possible. The](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-32-320.jpg)
![Labels and Probes for Live Cell Imaging 33
Fig. 2.9. Representative Zn2+
selective sensors. ZP2 and ZS5 are intensity based turn-on probes with sub nanomolar and
micromolar Zn2+
affinities, respectively. ZNP1 is a ratiometric sensor with dual emission at 545 and 624 nm.
from the low nM to M (Fig. 2.9) (86). One of these low-affinity
probes, ZS5 was used to visualize glutamate-mediated Zn(II)
uptake in dendrites and Zn(II) release resulting from nitrosative
stress (86). As with Lippard, Nagano has focused on design of flu-
orescent Zn2+
sensors based on the fluorescein scaffold. Probes of
the ZnAF family have low fluorescence background and strong
activation of up to 69-fold upon Zn2+
coordination (87, 88).
These sensors have been used to visualize Zn2+
release in the rat
hippocampus (88) and to monitor presynaptic Zn2+
pools (89).
Systematic modification of the dipicolylamine chelating moiety on
these probes has enabled preparation of ZnAF probes with Kd val-
ues ranging from 2.7 nM to 600 M (90). The ratiometric Zn2+
probes FuraZin and IndoZin, which are based on the related Fura
and Indo Ca2+
sensors, (91) were used to monitor intracellular
zinc uptake (92). Both FuraZin and IndoZin are excited at short
wavelength (400 nm) (91). To minimize potential phototoxic
effects of UV excitation, long-wavelength ratiometric probes such
as Zin-naphthopyr-1 (ZNP1), which has a 0.55 nM Zn2+
affin-
ity, were designed (Fig. 2.9) (93). The ZNP1 probe has dual
emission at 545 and 624 nm, where increasing [Zn2+
] induces a
dramatic increase in the 624-nm emission signal. The diacetate
derivative of this probe is membrane permeable and was used to
image release of Zn2+
from COS-7 cells in real time (93). A NIR-
emitting ratiometric probe, DIPCY, is based on a carbocyanine
fluorophore scaffold (94) This probe, which has a Zn2+
Kd of 98
nM, displays an approximate 50 nm red-shift in its absorbance
spectrum upon binding zinc.
Ion selective probes for H+
are one of the oldest and most
studied classes of ion sensors. Their development and use has been
vital for investigation of pH changes in the endosomal/lysosomal
system. Furthermore, disruption of acid/base homeostasis is asso-
ciated with the pathophysiology of diseases such as cancer, cystic
fibrosis, and immune dysfunction (95–98). Many fluorophores](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-46-320.jpg)
![gentlemen of England! Amid all the advantages and recreations
which have been pointed out in the preceding chapters as
surrounding the country life of modern England, that of scientific
farming is certainly one of the greatest. It is a pursuit full of interest
and variety, at once natural, philosophical, and dignified. It is difficult
to imagine a man of wealth and education more usefully or
honourably employed than in directing the culture and improvement
of his estate. Agriculture is now become, indeed, as Cicero termed it
in his day, “the nearest of all employments to the purely
philosophical kind.” It is a science which requires a first-rate
education to prosecute it to its full capability, to make the other arts
and sciences of modern times bear upon it, and co-operate with it,
so as to add something to its progression, or even to apply
beneficially the knowledge of its already established principles and
practices.[1] It is no longer an occupation which requires a man to
forego the refined pleasures of society, to bury himself amid woods
and wildernesses in some obscure hamlet far from the enjoyments
and intelligence of the world. As we have already seen, locate
himself where he will in these islands, the arts, the elegances, the
news and knowledge of civilized life, will penetrate to him by swift
agencies, and give him all the real advantages of the city in the
peace and fulness of his retirement. And what a noble art is
agriculture now become! Look at the manner it is now practised by
the most skilful of its professors. Let any one just turn over the
leaves of Mr. Loudon’s Encyclopædia of Agriculture, and trace the
progress of its implements only, from the plough of the ancients in
the shape of a mere pick, to the almost endless machines which the
active brains of men and their advancing knowledge of mechanics
have given to the scientific farmer. Let any one turn to the list of
engravings of farming apparatus in the same excellent work,
amounting to about 300, and he will obtain some idea of the amount
of science and invention now devoted to the use of the agriculturist.
There are no men who have availed themselves of the progress of
the arts and of general knowledge more than they. Mechanics,
chemistry, hydraulics, steam, all have been seized upon, to develope
the principles, or facilitate the operations of agriculture. Within the](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-76-320.jpg)
![[1]
weald of Kent, land which produced only a rental of five shillings an
acre, has been raised by this process to five-and-twenty. And all
these objects have been watched over, canvassed, and stimulated by
the establishment of agricultural societies, agricultural journals and
newspapers, and ploughing matches. Agricultural associations are
now to be found in almost every county, and in different districts of
the same county, which offer premiums on the best specimens of
horses, cattle, and sheep; the best ploughing, and the most steady
and industrious farm and household servants. It is a new feature in
rural life, to see the whole farming population of a district hastening
on a given day, gentlemen, farmers, and farm-servants all in their
best array, to some one spot where the cattle are shewn, the
ploughing is done, the prizes are awarded by umpires chosen from
the most skilful, and the different parties then going to a good
dinner, and a long talk and hearty toasting of all the interests of
agriculture.
This education is now likely to be extended to the great body of
farmers. In Ireland, at Templemoyle, a college is established where the
sons of farmers are instructed in every branch of science which can
enable them to pursue agriculture successfully, while they daily work
certain hours on the farm attached, thus making a familiar practical
acquaintance with all the best processes of cultivation under the ablest
professors. Similar colleges are also contemplated for England.
It is really too, as curious to see on our scientific farms the vast
variety of implements and machines which these causes have
produced;—ploughs—about a dozen and a half swing-ploughs, and
upwards of a dozen wheel-ploughs of different constructions, and by
different patentees; harrows, drills, cultivators. Every species of soil
and crop has its peculiar apparatus; in the field and the farm-yard;
for getting seed into the ground, clearing and dressing when there,
for thrashing it out and cleaning it for market; for sowing peas,
beans, turnips, carrots, parsnips, etc., for chopping, slicing, and
preparing them for cattle; their machines for tedding hay, for
stacking it with least possible risk, for cutting and steaming it; for
ploughing up weeds, ploughing up moorlands, and even roads; for
reaping by wholesale, and raking by wholesale; for tapping deep](https://image.slidesharecdn.com/95068-250411012634-8fe2a52f/85/Live-Cell-Imaging-Methods-and-Protocols-1st-Edition-Martin-Oheim-Auth-78-320.jpg)
Live Cell Imaging Methods and Protocols 1st Edition Martin Oheim (Auth.)
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- 6. METHODS IN MOLECULAR BIOLOGY TM Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651
- 7. Live Cell Imaging Methods and Protocols Edited by Dmitri B. Papkovsky University CollegeCork,Cork,Ireland
- 8. Editor Dmitri B. Papkovsky Department of Biochemistry University College Cork Cavanagh Pharmacy Bldg. College Road Cork Ireland d.papkovsky@ucc.ie ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-403-6 e-ISBN 978-1-60761-404-3 DOI 10.1007/978-1-60761-404-3 Library of Congress Control Number: 2009939132 © Humana Press, a part of Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper springer.com
- 9. Preface Live cell imaging has now become a routine tool in biomedical and life science research. It is hard to imagine an active academic research department, pharmaceutical or biotech- nology company without access to this technology and without using it on a regular basis. Over the last decade, major progress in this area has been achieved, making this core biochemical, cell and molecular biology techniques even more versatile, affordable, and mature. On the other hand, we continue witnessing numerous new, breakthrough developments which advance this technology even further, extending its capabilities and measurement standards. A variety of advanced-imaging methodologies, probe chemistries, experimental procedures, dedicated instruments, integrated systems, and a large number of new applications have come to the fore very recently. One can mention, for example, ultra-high resolution methods breaking the canonical diffraction limits, multi-photon exci- tation imaging and sample manipulation (e.g., (un)caging, permeabilization), new chemi- cally and genetically engineered probes for key markers and parameters of cellular function, multi-color imaging, specialized detection formats, custom-built systems employing new optoelectronics and engineering solutions, user-friendly multi-mode microscopes, soft- ware, and data analysis algorithms. All this provide unprecedented opportunities for the real-time investigation of live objects, including individual cells, sub-cellular organelles, and even individual molecules, with high level of detail and information content. Being until recently a privilege of large institutions and centralized facilities, live cell imaging sys- tems are now spreading into small labs, while sophisticated high content imaging stations are being deployed to screening labs. At the same time, the wide and ever increasing range of imaging techniques and appli- cations necessitates regular updates for existing users as well as an up-to-date introduc- tion and some general guidance for newcomers to this area. This volume of the Meth- ods in Molecular Biology series provides a comprehensive compendium of experimental approaches to live cell imaging in the form of several overview chapters followed by rep- resentative examples and case studies covering different aspects of the methodology. The 21 chapters of this volume are prepared by leaders in these fields, and the outstanding contribution of the authors is gratefully acknowledged. The book provides a range of state-of-the-art protocols extensively validated in complex biological studies. It highlights new experimental and instrumental opportunities and helps researchers to select appropri- ate imaging methods for their specific biological questions and measurement tasks. Each method also highlights the potential challenges and experimental artefacts which are likely to appear and which unfortunately are still not very uncommon. We believe that this vol- ume will contribute to the further development and dissemination of this fundamentally important technology which spans across many disciplines including molecular and cell biology, chemistry, physics, optics, engineering, cell physiology, and medicine. Dmitri B. Papkovsky v
- 10. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I GENERAL PRINCIPLES AND OVERVIEW . . . . . . . . . . . . . . . . . . 1 1. Instrumentation for Live-Cell Imaging and Main Formats . . . . . . . . . . . . 3 Martin Oheim 2. Labels and Probes for Live Cell Imaging: Overview and Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Scott A. Hilderbrand 3. Live Cell Imaging: An Industrial Perspective . . . . . . . . . . . . . . . . . . . 47 Terry McCann PART II IMAGING TECHNIQUES, PROBES, AND APPLICATIONS . . . . . . . . . . 67 4. Design of Fluorescent Fusion Protein Probes . . . . . . . . . . . . . . . . . . . 69 Elizabeth Pham and Kevin Truong 5. Synthetic Fluorescent Probes for Imaging of Peroxynitrite and Hypochlorous Acid in Living Cells . . . . . . . . . . . . . . . . . . . . . . 93 Dan Yang, Zhen-Ning Sun, Tao Peng, Hua-Li Wang, Jian-Gang Shen, Yan Chen, and Paul Kwong-Hang Tam 6. Photo-Activatable Probes for the Analysis of Receptor Function in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Wen-Hong Li 7. The Application of Fluorescent Probes for the Analysis of Lipid Dynamics During Phagocytosis . . . . . . . . . . . . . . . . . . . . . 121 Ronald S. Flannagan and Sergio Grinstein 8. Imaging of Mitotic Cell Division and Apoptotic Intra-Nuclear Processes in Multicolor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Kenji Sugimoto and Shigenobu Tone 9. Manipulation of Neutrophil-Like HL-60 Cells for the Study of Directed Cell Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Arthur Millius and Orion D. Weiner 10. A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy . . . 159 Hae Won Sohn, Pavel Tolar, Joseph Brzostowski, and Susan K. Pierce 11. Sample Preparation for STED Microscopy . . . . . . . . . . . . . . . . . . . . 185 Christian A. Wurm, Daniel Neumann, Roman Schmidt, Alexander Egner, and Stefan Jakobs vii
- 11. viii Contents 12. Two-Photon Permeabilization and Calcium Measurements in Cellular Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Oleg Gerasimenko and Julia Gerasimenko 13. Imaging and Analysis of Three-Dimensional Cell Culture Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Benedikt W. Graf and Stephen A. Boppart 14. Long-Term Imaging in Microfluidic Devices . . . . . . . . . . . . . . . . . . . 229 Gilles Charvin, Catherine Oikonomou, and Frederick Cross 15. Monitoring of Cellular Responses to Hypoxia . . . . . . . . . . . . . . . . . . 243 Christoph Wotzlaw and Joachim Fandrey 16. Imaging of Cellular Oxygen and Analysis of Metabolic Responses of Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Andreas Fercher, Tomas C. O’Riordan, Alexander V. Zhdanov, Ruslan I. Dmitriev, and Dmitri B. Papkovsky 17. Analysis of Mitochondrial pH and Ion Concentrations . . . . . . . . . . . . . . 275 Martin vandeVen, Corina Balut, Szilvia Baron, Ilse Smets, Paul Steels, and Marcel Ameloot 18. Live Cell Imaging Analysis of Receptor Function . . . . . . . . . . . . . . . . . 311 Daniel C. Worth and Maddy Parsons 19. Subcellular Dynamic Imaging of Protein–Protein Interactions in Live Cells by Bioluminescence Resonance Energy Transfer . . . . . . . . . . . . . . 325 Julie Perroy 20. Quantitative Analysis of Membrane Potentials . . . . . . . . . . . . . . . . . . 335 Manus W. Ward 21. Image Correlation Spectroscopy to Define Membrane Dynamics . . . . . . . . . 353 Jeremy Bonor and Anja Nohe Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
- 12. Contributors MARCEL AMELOOT • Cell Physiology Group, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium CORINA BALUT • Cell Biology and Physiology Department, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA SZILVIA BARON • Laboratory of Ca2+ -transport ATPases, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Belgium JEREMY BONOR • Department of Biological Sciences, University of Delaware, Newark, DE, USA STEPHEN A. BOPPART • Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA JOSEPH BRZOSTOWSKI • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA GILLES CHARVIN • Laboratoire Joliot-Curie & Laboratoire de Physique, Ecole Normale Supérieure, Lyon, France; The Rockefeller University, New York, NY, USA YAN CHEN • Department of Surgery, The University of Hong Kong, Hong Kong, P. R. China FREDERICK CROSS • The Rockefeller University, New York, NY, USA RUSLAN I. DMITRIEV • Biochemistry Department, University College Cork, Cork, Ireland ALEXANDER EGNER • Department of NanoBiophotonics, Max Planck Institute for Bio- physical Chemistry, Goettingen, Germany JOACHIM FANDREY • Institut für Physiologie, Universität Duisburg-Essen, Essen, Germany ANDREAS FERCHER • Biochemistry Department, University College Cork, Cork, Ireland RONALD S. FLANNAGAN • Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada JULIA GERASIMENKO • Department of Physiology, Biomedical School, University of Liverpool, Liverpool, UK OLEG GERASIMENKO • Department of Physiology, Biomedical School, University of Liverpool, Liverpool, UK BENEDIKT W. GRAF • Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA SERGIO GRINSTEIN • Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Biochemistry and Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada SCOTT A. HILDERBRAND • Center for Molecular Imaging Research, Massachusetts Gen- eral Hospital/Harvard Medical School, Charlestown, MA, USA STEFAN JAKOBS • Mitochondrial Structure and Dynamics/Department of NanoBiopho- tonics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany WEN-HONG LI • Departments of Cell Biology and Biochemistry, University of Texas South- western Medical Center, Dallas, TX, USA ix
- 13. x Contributors TERRY MCCANN • TJM Consultancy, Kent, UK ARTHUR MILLIUS • Cardiovascular Research Institute and Department of Biochemistry, University of California, San Francisco, CA, USA DANIEL NEUMANN • Mitochondrial Structure and Dynamics/Department of NanoBio- photonics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany ANJA NOHE • Department of Biological Sciences, University of Delaware, Newark, DE, USA MARTIN OHEIM • INSERM, U603, Paris, France; CNRS, UMR8154, Paris, France; Laboratory of Neurophysiology and New Microscopies, University Paris Descartes, Paris, France CATHERINE OIKONOMOU • The Rockefeller University, New York, NY, USA TOMAS C. O’RIORDAN • Luxcel Biosciences Ltd., BioTransfer Unit, UCC, Cork, Ireland DMITRI B. PAPKOVSKY • Biochemistry Department, University College Cork, Cork, Ireland MADDY PARSONS • Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK TAO PENG • Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China JULIE PERROY • Functional Genomic Institute, Department of Neurobiology, Unité mixte de recherche 5203 Centre National de la Recherche Scientifique, Unité 661 Institut National de la Santé et de la Recherche Médicale, Université Montpellier I & II, Montpellier, France ELIZABETH PHAM • Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada SUSAN K. PIERCE • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA ROMAN SCHMIDT • Department of NanoBiophotonics, Max Planck Institute for Biophysi- cal Chemistry, Goettingen, Germany JIAN-GANG SHEN • School of Chinese Medicine, The University of Hong Kong, Hong Kong, P. R. China ILSE SMETS • Department PHL-Bio, PHL University College, Diepenbeek, Belgium HAE WON SOHN • Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA PAUL STEELS • Cell Physiology Group, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium KENJI SUGIMOTO • Live Cell Imaging Institute, Osaka Prefecture University, Sakai, Osaka, Japan; Laboratory of Applied Molecular Biology, Division of Bioscience and Informatics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan ZHEN-NING SUN • Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China PAUL KWONG-HANG TAM • Department of Surgery, The University of Hong Kong, Hong Kong, P. R. China PAVEL TOLAR • Laboratory of Immunogenetics, National Institute of Allergy and Infec- tious Diseases, National Institutes of Health, Rockville, MD, USA SHIGENOBU TONE • Department of Biochemistry, Kawasaki Medical School, Okayama, Japan
- 14. Contributors xi KEVIN TRUONG • Institute of Biomaterials and Biomedical Engineering and Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, Toronto, ON, Canada MARTIN VANDEVEN • Cell Physiology Group, Biomedical Research Institute, Hasselt University Diepenbeek, Belgium HUA-LI WANG • Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China MANUS W. WARD • Department of Physiology and Medical Physics, Royal College of Sur- geons in Ireland, Dublin, Ireland ORION D. WEINER • Cardiovascular Research Institute and Department of Biochemistry, University of California, San Francisco, CA, USA DANIEL C. WORTH • Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK CHRISTOPH WOTZLAW • Institut für Physiologie, Universität Duisburg-Essen, Essen, Germany CHRISTIAN A. WURM • Mitochondrial Structure and Dynamics/Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany DAN YANG • Department of Chemistry, The University of Hong Kong, Hong Kong, P. R. China ALEXANDER V. ZHDANOV • Biochemistry Department, University College Cork, Cork, Ireland
- 15. Part I General Principles and Overview
- 16. Chapter 1 Instrumentation for Live-Cell Imaging and Main Formats Martin Oheim Abstract Unlike immunofluorescence confocal microscopy of fixed samples or microscopic surface analysis in mate- rial sciences that both involve largely indestructible samples, life-cell imaging focuses on live cells. Imag- ing live specimen is by definition minimally invasive imaging, and photon efficiency is the primordial concern, even before issues of spatial, temporal or, spectral resolution, of acquisition speed and image contrast come in. Beyond alerting the reader that good live-cell images are often not the crisp showcase images that you know from the front page, this chapter is concerned with providing a fresh look on one of the routine instruments in modern biological research. Irrespective of whether you are a young researcher setting up your own lab or a senior investigator choosing equipment for a new project, at some stage you will most likely face decision making on what (fluorescence) imaging set-up to buy. In as much as this choice is about a long-lived and often relatively costly piece of equipment and, more importantly, impacts on your future experimental program, this choice can be a tricky one. It involves considering a multitude of parameters, some of which are discussed here. Key words: Fluorescence, live-cell imaging, microscopy, instrumentation. 1. Introduction Fluorescence microscopy has evolved from an add-on contrast mode of the laboratory light microscope to a puzzling mul- titude of formats probing different aspects of molecular fluo- rophores. Classically requiring nothing else but a bright white light source for wide-field illumination (often termed a “burner”), a set of fluorophore-specific filters housed and purchased as a pre- assembled “cube,” and an imaging detector (“camera”), the rapid technological evolution of scientific instruments has involved vir- tually all elements of the fluorescence microscope. D.B. Papkovsky (ed.), Live Cell Imaging, Methods in Molecular Biology 591, DOI 10.1007/978-1-60761-404-3 1, © Humana Press, a part of Springer Science+Business Media, LLC 2010 3
- 17. 4 Oheim Fundamental choices for the user concern the illumina- tion source, where arc-lamps are increasingly being replaced by lasers or high-power light-emitting diodes (LEDs) as discrete- wave-band illumination devices, pulsed or continuous-wave exci- tation, point scanning vs. whole-field excitation, filter-based or dispersion-based fluorescence band selection, integrating vs. photon-counting detectors, multi-channel or spectral detection, intensity of lifetime detection, to name only a few. To these options concerning instrumentation add those coming from the rapid progress in the synthesis and generation of molecular fluo- rophores, photolabile caged compounds, fluorescent and photo- switchable proteins, and photoactivated ion channels that often call for specific add-ons and imaging modalities. Moreover, the detection of intrinsic signals (autofluorescence, scattered light, higher harmonic generation) offers interesting alternatives to conventional fluorescence imaging depending on the introduc- tion of exogenous probes. For a novice, it might appear difficult to navigate through this diversity of instrumentation, formats, and probes and to have an educated choice among the variety of equipment or software available. This chapter, without attempting to be complete, is meant to provide the groundwork for choosing and evaluating instrumentation for live-cell imaging. Emphasis is on principles and constraints imposed by the different techniques rather than on a detailed discussion of specific equipment. 1.1. Further Reading and Web Resources It is beyond the scope of this introduction to provide a detailed discussion of the ever increasing number of different formats of fluorescence microscopy. Fluorescence microscopy in its many variants is a standard theme in undergraduate and graduate courses, and a number of excellent reviews and textbooks are devoted to this subject; see below for a selection. To these, we have added the online resources provided by the different scien- tific societies as well as companies. We also alert the reader to the many excellent hands-on training courses that are held each summer and which – at least for the more prestigious ones – com- bine excellent theoretical training with the possibility to get your hands on the most recent pieces of equipment and thus provide valuable information before decision making about which piece of equipment to get for your own lab. Other important sources of first-hand information are the numerous cost-free optics and photonics journals. 1.2. Selected Fluorescence Textbooks Although by no means complete, these recent (re-)editions of classic books provide an in-depth coverage of many aspects of fluorescence microscopy techniques, with a specific emphasis on biological and live-cell imaging.
- 18. Imaging Instrumentation and Formats 5 1. Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Springer, Heidelberg, New York, 3rd edition, 2006 2. Pawley, J. B. (ed.), Handbook of Confocal Microscopy, Springer, Heidelberg, New York, 3rd edition, 2006 3. Goldman, R. D. and Spector, D. L. (eds.) Live Cell Imaging – A Laboratory Manual. CSHL Press, Cold Spring Habor, 2005 4. Imaging in Neuroscience and Development – A Laboratory Manual. CSHL Press, Cold-Spring Habor, 2005 1.3. Web-Based Resources Almost all microscope suppliers now offer free online tuto- rials that cover many aspects of microscopy: resolution, con- trast generation, microscopic optics, and basics of fluorescence microscopy. They also point the reader toward related courses (often organized in partnership with the companies) and review articles. 1. Olympus Microscopy resource center: http://www. olympusmicro.com/primer/java/index.html 2. Zeiss Microscopy, http://www.zeiss.com/, and then link to “Technical Information” 3. Nikon Microscopy, http://www.microscopyu.com/ tutorials/, and, specifically on confocal microscopy, http://www.microscopyu.com/articles/confocal/ 4. Leica Microsystems, http://www.leica-microsystems.com/ website, then link to “Leica Scientific Forum” 5. Molecular Expressions Images from the Microscope, National High Magnetic Field Laboratory (NHMFL), Tal- lahassee, http://micro.magnet.fsu.edu/ 1.4. Courses There is an ever-increasing number of courses that permit both theoretical training and hands-on experience. Here are some of the better known ones: 1. Marine Biological Laboratory (MBL), Woods Hole, http://www.mbl.edu/education/ 2. Cold Spring Habor Laboratory, Cold Spring Habor, http://meetings.cshl.edu/courses.html 3. NIH Bio-trac courses, Bethesda, http://www.biotrac.com/ pages/courses.html 4. Live-Cell Microscopy Course, UBC, Vancouver, http://www.3dcourse.ubc.ca 5. Quantitative Fluorescence Microscopy Course, Mount Desert Island Biological Laboratory (MDIBL), http://www.cbi.pitt.edu/qfm/index.html
- 19. 6 Oheim 6. Marine Biological Association, Plymouth, The Microe- lectrode Techniques for Cell Physiology, http:// www.mba.ac.uk/events.php 7. European Molecular Biology Organization, Practical Courses, EMBO Course on Light Microscopy in Living Cells, Heidelberg, http://www.mba.ac.uk/events.php A more comprehensive list is found by linking to http://www.olympusfluoview.com/resources/courses.html 1.5. Free Photonics Journals Another valuable source of information for beginning as well as confirmed microscopists is freely available monthly journals. A particularity of the photonics market, there are quite a few of them. Although often in close (sometimes all-too-close) prox- imity with advertiser and manufacturer opinion, these publica- tions are a showcase of recent developments in optics, microscopy, and biophotonics. They provide up-to-date information on new equipment, notable technical achievements, and provide an excel- lent overview of trade fairs and meetings, both to come (which is good if you are to chose components for your microscope), or in the form of brief synopses, reviewing recent trends. While not replacing the academic literature, they certainly broaden your horizon and keep you connected to the often rapidly evolving technology at no extra cost. 1. Photonics Spectra 2. Europhotonics 3. Biophotonics International, all three from Photonics Media, Laurin Publishing, http://www.photonics.com 4. BioTechniques, from Informa Healthcare, http://www. biotechniques.com 5. Optics & Laser Europe, IOP Publishing, http:// www.optics.org 6. Photonik international (German) from AT-Fachverlag, http://www.photonik.de 7. Photoniques, de la Société Française d’Optique, http://www.photoniques.com 1.6. Keep Your Cells Alive When imaging live samples, the first question arising is how to use the limited photon budget without compromising sample viability. Careful controls should be made to develop a quan- titative notion of how the used intensities affect the biological process under study. The result is often surprising because pho- todamage starts gradually before obvious signs occur. For exam- ple, two studies investigating how ample was two-photon photo- damage when imaging intracellular free calcium ([Ca2+ ]i) (1, 2) found that the slope and kinetics of neuronal calcium signals were attenuated much earlier than electrophysiological signs of
- 20. Imaging Instrumentation and Formats 7 photodamage occurred. Thus, before starting your imaging, begin by thinking how you can • optimize the photon yield (i.e., the number of collected signal photons vs. photons injected into the sample), • avoid excitation wavelengths at which the sample absorption (and hence heating and photodamage) is strong, • minimize the applied dye concentration, • avoid producing crisp showcase images but aim for live cells, • repeat the same experiment using different excitation inten- sities and using different emission bands, • choose the imaging format that excels in your specific appli- cation. This last point is crucial because it involves a choice of equip- ment and may lead you to the conclusion that the ideal experi- ment is impossible with existing material and can only be realized through an external collaboration. Figure 1.1 provides a scheme (3) that helps rationalizing this decision making. Although over- simplistic, this scheme is useful because it points at the limita- tions of particular techniques and brings up useful parameters that should go into your consideration, e.g., background rejec- tion, optical sectioning, imaging speed, or penetration depth (to only name a few). 1.7. Do You Really Need a Microscope? Free yourself from the acquired wisdom that the choice is among the diverse upright and inverted scopes offered by the “big four,” Zeiss, Leica, Nikon, and Olympus, and that the decision is merely a question of preference, compatibility with existing equipment, or the best commercial offer. Instead, ask yourself the following questions: • Do I really need a microscope? • Do I need eyepieces, bulky microscope bodies, inaccessible and unchangeable intermediate optics, and a limited flexibil- ity governed by the elements to select from the suppliers’ catalogue? • You probably already have a good routine microscope for cell culture, patch-clamping, or checking immunofluorescence labeling before going to your facility’s confocal or routine imaging. But do you need to combine different imaging for- mats as expensive (and often sub-optimal) add-ons on the same instrument? • Or can you do better, with dedicated, small, and inexpensive set-ups? A number of companies now offer modular solutions that allow you to configure the microscope the best way according to your needs. These solutions provide an interesting alternative
- 21. 8 Oheim Start contrast ? Y TLM Y fluo ? N N N N N SHG, THG CARS near membrane? TIRFM Y deeper 100 µm ? 2PEF Y < 20 µm ? Y WFM video rate ? N CLSM 2D/3D ? Y Y SDC N line scan/ ROI CLSM Fig. 1.1. Abbreviations: Y/N – yes/no; TLM – transmitted-light microscopies; SHG, THG – second (third) harmonic generation; CARS – coherent anti-Stokes Raman scattering; TIRFM – total internal reflection fluorescence microscopy; WFM – wide-field microscopy; 2PEF – two-photon excitation fluorescence; CLSM –confocal laser scanning microscopy; ROI – region of interest; SDC – spinning-disk confocal. to the often somewhat finicky custom microscopes assembled from optical bench systems and also provide a means to gen- erate hybrids between commercial microscope components and custom optomechanics. All of these approaches have in common that they offer full control of what you put in your microscope and allow replacing specific components that become limiting in
- 22. Imaging Instrumentation and Formats 9 a given application. We later discuss two examples of such custom microscopes built in our own laboratory. Below, some of such modular microscopes the author is cur- rently aware of are listed. 1. Olympus BXFM series (part of the BX51/BX61 upright microscope series). Most components of this upright microscope are sold individually, permitting to build your own structures. 2. Zeiss Axio Scope 1.Vario, a more recent but similar, component-based upright and highly modular micro- scope originally designed for the material sciences, http://www.zeiss.de/C12567BE0045ACF1/Contents- Frame/8FE44E3197A08FEBC125742E005BD1E1. 3. Already somewhat more reductionist is the SliceScope from Scientifica. It is a commercial minimally stripped down microscope body (without eyepieces) for com- bined electrophysiology and DIC/fluorescence imaging, http://www.scientifica.uk.com. 4. A similar system, equipped with Dodt contrast, is avail- able from Siskiyou, http://www.siskiyou.com/imaging system mrk200-infrared-fluorescence.shtml. 5. TILL Photonics, offers a highly modular automated microscope that even goes below a bench or a robotic sample handler. Here the approach is rather to rethink the microscope body and to evolve in the direction of screening-by-imaging, http://www.till- photonics.com/Products/imic.php. With their now selling YANUS-4 laser scan head driven by all-digital smartmove boards, they equally offer a building block for constructing your own confocal or two-photon laser scanning micro- scope. 6. Somewhat more integrated, Prairie Technologies’ Ultima is a confocal attachment with (optionally) two sets of galvanotmetric mirrors or acousto-optic deflectors for combined imaging and uncaging, http://www.prairie- technologies.com/ultima.htm. 7. Becker & Hickl has a confocal scan head (DCS-120) and detector modules for both intensity and fluorescence life- time measurements, http://www.becker-hickl.de/. 8. Even further toward DIY, the opto-mecanical compo- nent supplier Linos (formerly Spinder und Hoyer) has its classical microbench, a 40 by 40 mm cage and rail system with components (eyepieces, revolver, lenses, C-mounts, apertures, ...) ready to build a microscope from scratch, http://www.linos.com/pages/home/shop- mechanik/banksysteme/mikrobank/.
- 23. 10 Oheim Also exists in a 20 × 20-mm nanobench version. Unfor- tunately, the future of the even larger macrobench (150 × 150 mm) is with a question mark. 9. AHF (http://ahf.de) has the missing part for the microbench: a 45◦ holder for a 1-mm 25 × 63-mm (Zeiss or Olympus) standard dichroic mirror. 10. Interestingly, Thorlabs offers a fair number of cage system components that are largely compatible with the Microbench, thus considerably enlarging the choice of optical components, http://www.thorlabs.com/ navigation.cfm?Guide ID=2002. 2. Instrumenta- tion 2.1. Understanding the Building Blocks of the Laboratory Microscope It is quite instructive to forget what you know about laboratory microscopes for a moment. Modern fluorescence microscopes are highly modular and can be thought of as a box with lots of arms sticking in and out (Fig. 1.2). Excitation arms can be different channels of epi-illumination, a laser injected through a side port for total internal fluorescence or photoswitching, a spinning-disc confocal attachment, or a pulsed UV-lamp for flash-photolysis. For a given excitation channel i and fluorophore j, excita- tion is fully described in terms of the source spectral emission S(λ), transmission of the excitation filter EX(λ), and the reflectiv- ity diachroic beamsplitter (1–BS(λ)), which, for a give excitation channel, are multiplied along the excitation optical path: exi (λ) = S(λ) · EXi (λ) · (1 − BS(λ)) [1] combined with the sample molar extinction εj and absorbance spectrum Ej(λ) and integrated over λ to give an excitation spectral function: ξi j = dλ exi (λ) · E j (λ) · εj . [2] On the emission site, one proceeds analogously for each detection arm k by multiplying the fluorophore quantum yield and emission spectrum, spectral transmission of the dichroic and emission filters, and the detector spectral sensitivity: emk(λ) = BS(λ) · EMk(λ) · D(λ). [3] Upon integration, ξ jk = dλφ · Fj (λ) · emk(λ) [4]
- 24. Imaging Instrumentation and Formats 11 Finally, the product of eqs. [2] and [1.4], ξi jk ≡ ξi j ξ jk [5] measures the signal of one mole/l of fluorophore j viewed through channel k upon excitation in channel i, and similarly for all permutations ijk. Optimizing the photon yield now consists in maximizing the detection spectral function, while balancing the excitation spectral function with that of other fluorophores present in the sample. This balancing can be achieved not only by choosing appro- priate filters but also by considering the source and detector spec- tra and the microscope intermediate optics and objective lens. Their transmission can conveniently be combined with that of the beamsplitter, thereby accounting for the double passage of excitation and emission light through the microscope. The following list provides a quick overview of the principal choices available and briefly discusses their respective advantages and disadvantages with respect to the scheme shown in Fig. 1.2. D(λ) S(λ) EM(λ) EX(λ) BS(λ) M excitation channel(s) N fluorophore(s) L detection channel(s) S(λ) · EX1(λ) · (1–BS(λ)) E1(λ) · (ε1φ1) · F1(λ) E2(λ) · (ε2φ2) · F2(λ) S(λ) · EX2(λ) · (1–BS(λ)) BS(λ) · EM2(λ) · D(λ) BS(λ) · EM1(λ) · D(λ) … … … a b Fig. 1.2. (a) Schematic respresentation of an upright epifluorescence microscope. S – source; EX – excitation filter; BS – diachroic beamsplitter; EM – emission filter; D – detector. See main text for details. (b) Box plot of the different excitation and emission channels along with their spectral throughput, obtained by multiplying instrument and fluorophore spectral properties along the excitation and detection arms. See main text for details.
- 25. 12 Oheim Light sources: the choice is no longer only between Hg- and Xe-burners but involves a large variety of sources including arc-lamp based monochromators, lasers (gas, diode-pumped solid state, DPSS), laser diodes, white light and color LEDs, and pho- tonic crystal fibers for supercontinuum generation. These vary in • wavelength, tuning range, bandwidth and power, • luminous density, • noise and long-term stability, • cost, • coherence, polarization, • pulsed vs. continous-wave (CV) operation. Excitation filters: typically, band-pass filters are used to nar- row down the source spectrum and select specific wavelength bands, clean up laser lines, attenuate illumination intensity, or select a polarization direction. Main choices are between hard- and soft-coated interference filters as well as holographic notch filters. Important selection criteria include the following: • their in-band transmission and off-band rejection (optical density), • bandwidth or line-width, • tolerance to high illuminating intensities (burnout, hole- burning). A similar reasoning must be made for the dichroic mirror that, for the more common long-pass, separates excitation and emission light by reflecting the excitation light onto the sample and transmitting the collected fluorescence. Chief parameters to consider in addition to center wavelength and steepness are as follows: • Spectral holes: many dichroics perform nicely close to the transition wavelength, but display large variations and spectral holes at remote wavelength. “Extended range” (“XR”) dichroics can be a solution, as are the new hard- coated filters that typically outperform the older soft coatings. • Transmitted excitation light. Although often not a problem, low-light applications can suffer from the residual transmit- ted light that can be orders of magnitude more intense than the collected fluorescence. Stacking dichroics or matched long-pass filters can be an answer. • Finally, particularly for multi-color scanning microscopies, the angle-dependence of the cut-on of the dichroic can result in surprising chromatic changes depending on whether paraxial regions or the periphery of the field-of-view is imaged. Typically, the cut-on wavelength changes as
- 26. Imaging Instrumentation and Formats 13 λ(θ) = λ0 1 − (sinθ/neff)2, [6] For angles θ increasing beyond 45◦ , neff is determined by the dichroic of the order of 1.7–1.86 for most of the coatings, but varies for p- or s-polarized light so that their average must typically be considered for the collected fluorescence. The microscope intermediate optical path (tube lens, scan lens, epi-illuminator, projection lenses in the case of compound mag- nifiers) and the objective are often not considered in detail, until unanticipated losses occur. For example, both UV and near-IR transmission become a problem, e.g., in fura-2 Ca2+ imaging, flash photolysis, or photoactivation, as well as two- or three- photon microscopy using far-IR excitation. • UV transmission. With the increasing demand for highly corrected objectives (and tube lenses), high refractive index glasses and optical cement are often used chromatic correc- tions that limit UV transmission. Also, with microelectron- ics getting smaller and smaller, the formerly used UV-light inspection techniques often fail because of resolution limita- tions. Thus, dedicated UV-transmitting optics is getting rarer and rarer. • Another often overlooked aspect is limiting intermediate apertures (filter cubes, lenses, irises) that reduce the col- lected light fraction of scattered fluorescence photons in two-photon microscopy. While scattered photons are usu- ally rejected in fluorescence imaging (because they are not assigned to the pixel of their origin and hence blur the image) they constitute useful signal in 2PEF microscopy that seeks to optimize the light collection. Thus, keeping the product of d sin(θeff) constant is of crucial importance for not using light. Here, d is the size of the imaged spot and θeff is the half-angle of the effective numerical aperture. Much of the same reasoning is true for choosing and optimiz- ing detectors. Know your microscope is the rule toward getting optimal results. 2.2. The Performance Triangle Irrespective of all these parameters, you can only distribute your collected photons once. Thus, whenever you take an image of a live cell, you take from the budget of photons that your sam- ple emits before irreversibly undergoing photodamage and pho- todestruction. If you invest them into higher spatial resolution, higher temporal resolution or larger image contrast (signal-to- noise ratio) is your choice. And it is often a difficult one. Resolution vs. magnification. On most available microscopes, resolution is diffraction-limited. Thus, the smallest distance that two objects can be close by and still be detected as two is defined by the numerical aperture of the objective, according to Abbe’s
- 27. 14 Oheim law. Importantly, the detector spatial resolution (i.e., the pixel size in an imaging detector, or the scan angle for a laser-scanning microscope) must be adapted, according to Nyquist’s sampling theorem: two picture elements (pixels) per resel (resolution ele- ment), 0.61λ/(NA). Smaller pixels do not enhance resolution but increase photobleaching by void oversampling. Superresolution, i.e., imaging beyond the diffraction limit has attracted wide interest over the last years and extensive reviews have been published. By narrowing down the fluorescence excita- tion volume (through stimulated emission depletion, STED, or structured illumination and image reconstruction methods) or by sequentially imaging individual fluorophores and reconstruct- ing the image from the sum projection (STORM, PALM, and its variants) high spatial frequency information can be obtained. But again, superresolution translates into sampling at higher spa- tial frequencies, so that at a constant photo budget, either the dye concentration in the sample must be increased or the field-of-view must be reduced. Also, some super-resolution techniques require sample pre-bleaching, photoactivation, or the STED beam in addition to the conventional excitation light. It is safe to say that for many of these exciting developments, a critical evaluation of the photodamage resulting in the live samples still has to be done. Image contrast comes from the number of meaningful sig- nal photons over the unwanted background in a given fluores- cence detection channel. Thus, spectral considerations directly come into play. If contrast is generated by splitting up the sig- nal in many different spectral channels, then each of these chan- nels will contribute to the noise and the signal-to-noise ratio will inevitably drop. Therefore, “multi-”spectral imaging is generally preferable over “hyper-”spectral imaging and a small number of detection channels followed by spectral unmixing outperforms full-blown spectral images (4, 5). Similarly, contrast in a given spectral bin can of course be increased by cranking the laser power up, but this again increases photobleaching and tears from the available photon budget. Therefore, it is useful to keep the “per- formance triangle” of fluorescence microscopy (see Fig. 1.3) in mind, any improvement in one of the image parameters comes at the expense of the others. 2.3. Additional Considerations • Long-term observation of live samples critically relies on con- stant observation conditions, both in terms of the instrument (i.e., maintaining the focal stability and minimizing thermal drift) and of the biological sample (control of physiologi- cal temperature, ambient CO2, and humidity levels). Due to condensation, placing the entire microscope in an incubator is not preferred, but many suppliers offer small-on stage or plexiglass-box incubators that can be fitted to many routine microscopes.
- 28. Imaging Instrumentation and Formats 15 spatial resolution contrast temporal resolution Fig. 1.3. The performance triangle. Distributing a constant photon budget into any of these three imaging parameters inevitably reduces the other two. • Deciding between a motorized vs. manual set-up is not only a question of price and convenience. Many microscopists like the manual and thus more direct focus and stage con- trol, e.g., when placing recording electrodes or local perfu- sion systems under visual control. On the other hand, many motorized systems now come with a fairly well-developed software, allowing the user to generate look-up tables of stage positions, objective z-positions or even automated fol- low software that keeps patch pipettes already placed above the sample plane in the field-of-view whilst searching for the cell to record from. • High-throughput microscopy is increasingly becoming an option through the ongoing integration of machine vision, robotics, microfluidics, and automated analysis software. Sev- eral commercial systems are available, albeit at high cost. • Shared set-ups vs. single-user set-ups. Perform a realistic eval- uation of beamtime: which fraction of your experiment time is effectively being used for imaging? Which part is devoted to preparing and installing the sample? Which steps could equally be performed elsewhere to free precious beamtime? Most laboratory microscopes are under-used, but multi-user set-ups require clear shared responsibilities, agreed-on stan- dard operation protocols, and an efficient communication among users. Otherwise, the gain in instrument use will eas- ily be eaten up by problem solving and conflicts of unhappy experimenters. • Multi-functionality vs. dedicated set-up. Beyond budgetary and space constraints, this often is a question of the type of experiments you have in mind. All too complicated micro- scopes are expensive, error-prone, and rarely all contrast modes are being used in the same experiment. Thus, “small is beautiful” is a guideline that more often than not gives good returns, particularly when set-ups are shared or even open to external users. Trained personnel and regular maintenance will make all the difference.
- 29. 16 Oheim • Optimal optical performance in a given imaging format often involves custom equipment or add-ons to commercial micro- scopes. Typically being fairly labor-intensive, alignment sen- sitive, and often run with custom software that resembles beta-versions, these “expert systems” are less user-friendly but outperform standard equipment. Many recent imag- ing formats, including light-sheet based illumination, HILO (6, 7), but also versatile STED or 2PEF microscopes, still require building your own setup. At the same time, instru- ment development and building is typically longer than hoped for, so that the researcher has to evaluate the need for quick results against instrument performance. • Laser safety is obviously a concern, particularly with home- built apparatus. While interlocks, beam stops, and protective shutters are mandatory in commercial microscopes, custom set-ups are often more reminiscent of open optical bench sys- tems and do not comply with legal guidelines. Hence, devel- opers and experimenters should stay in close contact and new users should be briefed about risks. 3. Concluding Remarks Neither does this introduction replace a careful reading of the original papers describing different imaging formats nor does it replace making your own experience with the equipment you bought. But it alerts the reader to consider some parameters that are not so obvious when looking at microscope brochures and reading the often very condensed “materials and methods” sec- tions. If there is a simple conclusion, then it is this: know your microscope. It pays for your research and your next budget. References 1. Hopt, A. and E. Neher, Highly nonlinear photodammage in two-photon fluorescence mi- croscopy. Biophys. J., 2001. 80(4): 2029–36. 2. Koester, H.J., et al., Ca2+ fluorescence imag- ing with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J., 1999. 77(4): 2226–36. 3. Frigault, M.M., et al., Live-cell imaging: tips and tools. Biophys. J., 2009. 96(3): Supple- ment 1, 30a. 4. Nadrigny, F., et al., Detecting fluorescent pro- tein expression and co-localization on single secretory vesicles with linear spectral unmixing. Eur. J. Biophys., 2006. 35(6): 533–47. 5. Neher, R.A. and E. Neher, Optimizing imag- ing parameters for the separation of multi- ple labels in a fluorescence image. J. Microsc., 2004. 213(1): 46–62. 6. Tokunaga, M., N. Imamoto, and K. Sakata- Sogawa, Highly inclined thin illumina- tion enables clear single-molecule imag- ing in cells. Nat. Methods, 2008. 5: 159–61. 7. van’t Hoff, M., V. de Sars, and M. Oheim, A programmable light engine for quan- titative single-molecule TIRF and HILO imaging. Opt. Express, 2008. 16(22): 18495–504.
- 30. Chapter 2 Labels and Probes for Live Cell Imaging: Overview and Selection Guide Scott A. Hilderbrand Abstract Fluorescence imaging is an important tool for molecular biology research. There is a wide array of flu- orescent labels and activatable probes available for investigation of biochemical processes at a molecular level in living cells. Given the large number of potential imaging agents and numerous variables that can impact the utility of these fluorescent materials for imaging, selection of the appropriate probes can be a difficult task. In this report an overview of fluorescent imaging agents and details on their optical and physical properties that can impact their function are presented. Key words: Fluorescence, fluorescent labels, fluorogenic probes, sensors, microscopy, imaging. 1. Introduction Fluorescence imaging is a vital tool for the investigation of bio- logical processes in the fields of cell, molecular, and systems biol- ogy. Its development has had a profound impact on our abil- ity to decipher how these systems function at the cellular and molecular level. The development of fluorescence microscopy as an investigative tool has its origins in the 1850s with the first descriptions of “refrangible radiations” from biological materials by George Stokes (1). These radiations were later named fluo- rescence. However, the use of fluorescence as a diagnostic tool in microscopy would remain undeveloped until the construction of the first UV light microscopes by August Köhler in 1904 (2). Not long after the work of Köhler, the first purpose-built fluores- cence microscopes were prepared, but it was not until the 1960s D.B. Papkovsky (ed.), Live Cell Imaging, Methods in Molecular Biology 591, DOI 10.1007/978-1-60761-404-3 2, © Humana Press, a part of Springer Science+Business Media, LLC 2010 17
- 31. 18 Hilderbrand that these instruments became commonplace. Some of the first fluorescence microscopy experiments focused on observation of the intrinsic fluorescence of the biological samples under inves- tigation (3). These investigations were invaluable for expanding our understanding of physiology, but they provided little insight on the function of biochemical and other physiological processes at a molecular level. For a more in depth investigation of these processes within the cell, a switch from intrinsic to extrinsic fluo- rophores is necessary. Today, numerous fluorescent materials are available for use in fluorescence microscopy. Fluorescent compounds suitable for live cell imaging can be divided into two broad categories: labels and responsive probes. Fluorescent labels are imaging agents whose fluorescence signal remains constant. Good labels are typified by stable optical prop- erties that do not vary significantly as a function of their local envi- ronment. These fluorescent species are often coupled with target- ing groups or have genetically controlled expression. Responsive probes do not rely on preferential uptake or targeting. These sen- sors rely on changes in fluorescence intensity, wavelength, or life- time for their function, and can be small molecule, polymer, or nanoparticle based. In this report we will provide an overview of current fluorescent labels and probes for use in live cell imaging of molecular processes. 2. Fluorescent Labels The first advances toward the development of modern fluores- cent labels are credited to the immunologist, Albert Coons in the 1940s. In his early research, he developed fluorescein isoth- iocyanate (FITC) (4), which remains one of the most ubiqui- tous fluorescent labels today, for coupling to antibodies targeted against pneumococcal bacteria. Today, targeted labels are among the most commonly employed fluorescent imaging agents. In addition to antibodies, targeting groups can be proteins, pep- tides, DNA aptamers, small molecule ligands, or stains for specific macromolecular structures. The emissive reporters in these labels can be fluorophores, fluorescent or bioluminescent proteins, or nanoparticles such as quantum dots. The efficacy of imaging with these compounds is dependent on their specific uptake, seques- tration, or expression at a subcellular level. 2.1. Small Molecule Fluorophores Prior to the development of FITC labels, a limited number of fluorophores with synthetic handles suitable for bioconjugation were available. Many of these early labels were based on dyes with fluorescence excitation in the UV (5). The fluorescence emission
- 32. Labels and Probes for Live Cell Imaging 19 signals from these dyes can be difficult to separate from tissue autofluorescence. In contrast, today there is a vast and often con- fusing array of fluorophore labels available to scientists. These flu- orophores span the optical spectrum from the UV to the visible and extend into the near infrared (6–12). Many of the currently available amine reactive fluorophores are summarized in Fig. 2.1 and the structures of some representative labels are shown in Fig. 2.2. Several factors must be considered in choosing the appropri- ate fluorophores for constructing effective imaging agents. These include method of attachment to the targeting group, excitation and emission wavelengths, brightness, hydrophilicity, and cost. There are many current chemistries available for the coupling of fluorescent labels to biomolecules and targeting groups (Fig. 2.3). The most frequently employed synthetic handle for biocon- jugation is the succinimidyl ester, which forms stable amide bonds after reaction with primary and secondary amines. The isothio- cyanate group may also be used for coupling to amines, generat- ing a thiourea linkage. In cases where reaction of a succinimidyl ester or isothiocyanate derivatized fluorophore with an amine is not feasible, additional coupling groups are available. Iodoac- etamide, maleimide, and dithiol-modified fluorophores are use- ful for covalent conjugation to thiols. Hydrazine and hydrazide modified dyes can be used for coupling to aldehydes and ketones, forming relatively stable hydrazone linkages. More recently, the development of bioorthogonal coupling schemes has attracted significant interest for preparation of fluorescent probes. Bioorthogonal couplings rely on use of reaction partners that display little or no reactivity with common biological materials. Two examples of these reactions are the Staudinger ligation (13) and the “click” reaction (14). The Staudinger ligation involves coupling of a methyl ester electrophilic trap with an azide to generate an amide linkage and one equivalent of N2. This reac- tion is mediated by oxidation of an adjacent phosphine. The click reaction is a copper(I) catalyzed [3+2] cycloaddition between an azide and an alkyne that results in the formation of a stable tri- azole product (14). This reaction has excellent potential for use in design of targeted fluorescent probes. However, there are only a few azide or alkyne modified dyes currently available for this reaction, most of which emit in the visible region. The poten- tial utility of the click reaction in biology suggests that in the coming years the selection of azide and alkyne modified dyes is likely to expand greatly. For example, recent efforts have yielded new, efficient synthetic routes to far-red/near infrared emitting cyanine dyes modified with either azide or alkyne groups, one example of which, CyAM-5 alkyne, is shown in Fig. 2.2 (15). Although highly selective, cytotoxic copper(I) is necessary for the traditional click coupling, and therefore direct use of this reaction in living biological systems has not been possible. The
- 33. 20 Hilderbrand Fig. 2.1. Commercial amine reactive fluorophore labels.
- 34. Labels and Probes for Live Cell Imaging 21 Fig. 2.2. Representative structures of fluorescent labels with emission in the blue (7-(diethylamino)coumarin-3-carboxylic acid NHS), green (BODIPY FL, FITC, and AF 488), orange (5-carboxy-tetramethylrhodamine NHS and Cy 3), far red (CyAM-5 alkyne), and near infrared (Cy 7). Fig. 2.3. Common coupling chemistries for attachment of fluorescent labels to targeting groups and biomolecules. issue of copper cytotoxicity in the click reaction has been over- come by Bertozzi and others via preparation of new ring- strained cyclooctyne derivatives that do not require a catalyst (16–18). The coupling of cyclooctyne containing fluorophores with azide-modified sugars has been demonstrated for imaging
- 35. 22 Hilderbrand of surface glycosylation in live cells (16) and zebra fish embryos (19). Another bioorthogonal conjugation strategy compatible with live cells was reported in 2008 (20, 21). This coupling scheme involves use of an inverse-electron demand Diels–Alder cycloaddition between a modified tetrazine and a norbornene dienophile (20). The tetrazine-based coupling shows excellent selectivity in biological media and was used to label SKBR3 breast cancer cells that were pre-treated with norbornene mod- ified trastuzumab (Fig. 2.4). The availability of labels for use with classical and bioorthogonal coupling reactions provides a wide selection of methods for attachment of fluorescent reporters to biological targets. The choice of coupling chemistry will be dependent on the specific reactive chemical groups available on the targeting molecule such as amines, thiols, or ketones. When the coupling or labeling reaction must be performed in a bio- logical environment in the presence of live cells, the copper-free click reaction, Staudinger ligation, or the tetrazine cycloaddition reactions are appropriate conjugation methods. Fig. 2.4. Pre-targeting of GFP expressing SKBR3 human breast cancer cells with norbornene modified trastuzumab antibodies followed by addition of tetrazine-VT680, which covalently couples to the norbornene groups in an inverse electron demand Diels-Alder cycloaddition (panel A). Panel B shows confocal microscopy images of the cells after pre-targeting and VT-680 treatment in the GFP channel (left), VT680 channel (center), and the merged image (right). Coupling chemistries have been used to prepare a wide array of imaging agents utilizing antibodies, aptamers, peptides, and small molecules. For example, anti-human epidermal growth fac- tor 2 (HER2) antibodies conjugated with IRDye 800 were used to show antibody binding to HER2 expressing SKBR3 breast cancer cells and for in vivo fluorescence imaging in a mouse
- 36. Labels and Probes for Live Cell Imaging 23 model (22). DNA aptamers have also been used to target tumor cells (23). In much the same way, peptides have been coupled to a variety of fluorophores for preparation of several targeted imag- ing agents. This strategy has been widely used for targeting the ␣3 integrin cell adhesion molecule with the RGD peptide motif for investigation of cancer cells and tissues (24–27). Targeting approaches need not be limited to short peptides. Larger pep- tides and proteins may also be used for directed delivery of opti- cal reporters. Probes for selective imaging of epidermal growth factor receptor (EGFR) have been prepared via conjugation of Cy5.5 fluorophores to the 6-kDa epidermal growth factor pro- tein. This probe was demonstrated to specifically home in on MDA-MB-468 cancer cells, which have high EGFR expression levels, but not to MDA-MB-435 cells which do not express EGFR (28). Small molecule based targeting strategies have also been employed through use of well-known bioactive small molecules such as folate (29) or through combinatorial approaches (30). Peptide-based targeting has been expanded to incorporate bacteriophage nanoparticles as multivalent peptide carriers. This allows for facile integration of peptide screening for the cellu- lar target of interest (via use of bacteriophage display libraries) with optical imaging and microscopy techniques. The M13 bac- teriophage, commonly used in bacteriophage screening, has ran- domized peptide libraries displayed on its pIII coat proteins. The bacteriophage particles also contain 2700 copies of the pVIII coat protein, which have their amino termini exposed to the sol- vent. These amine groups are available for bioconjugation to fluo- rophores. Therefore, once a phage clone specific for the receptor of interest is identified, it can be modified via standard succin- imide or isothiocyanate coupling procedures to prepare a fluores- cent targeted imaging probe. This strategy was first demonstrated in 2004 (31) and further expanded for other imaging applications (32–34). In addition to the bioconjugation strategy and selection of the targeting group, the optical properties of the fluorophore are another important factor in the design of targeted probes for live cell imaging. Although there are many fluorophores with excitation and emission in the UV, these fluorescent labels are not appropriate for certain imaging applications due to con- cerns regarding exposure of the cells to UV light, which may disrupt normal cell function. UV excitation may also result in higher background fluorescence signal from the sample, arising from the excitation of intrinsic biological fluorophores. Problems may occur with other common dyes, such fluorescein. Fluores- cein is a pH-sensitive dye with a fluorogenic pKa of 6.4; there- fore, fluorescent labels containing fluorescein may display dis- tinctly different fluorescence emission intensities depending upon the pH of their local environment. Consequently, for imaging
- 37. 24 Hilderbrand applications where the probe signal will be quantified, fluores- cein may not be suitable. Other fluorophores with similar excita- tion and emission such as BODIPY FL or AF488 (6), which is based on a pH-insensitive rhodamine scaffold, are more appropri- ate for use in experiments requiring probe quantification. In many applications, labels with fluorescence emission in the NIR are pre- ferred. NIR-emitting fluorophores are not susceptible to interfer- ence from biological autofluorescence and are directly translatable to many in vivo imaging applications due to the increased opti- cal transparency of biological tissue between ∼650 and 1000 nm (35). Fluorophores can show distinct changes in their fluores- cence intensity and/or fluorescence emission wavelength based on the polarity of their local environment. The fluorescence quan- tum yields and emission wavelengths of dansyl fluorophores are well known to vary with the polarity of the surrounding media. Solvent polarity-based changes in fluorescence emission wave- lengths and quantum yields can often be minimized by increas- ing the polarity of the fluorophore. Fluorophores that show little or no polarity-dependent changes on their optical properties tend to contain one or more solubilizing groups such as sulfonate or carboxylate moieties. For example, the optical properties of the near infrared emitting fluorophore, Cy5.5, which has four sul- fonate groups, are relatively insensitive to changes in the local microenvironment. Many fluorophores can be modified to act as optical switches that are activated by exposure to UV light. Several applications have made use of these photoactivatable or “caged” fluorophores. Caged fluorophores have been employed in dynamic imaging applications where specific temporal and spatial activation of a small population of fluorophore labels is required. These masked fluorophores, such as caged fluorescein, are prepared by reac- tion of the fluorophore with o-nitrobenzylbromide to form the non-fluorescent photoactivatable compound (36). The fluores- cence can be activated by irradiation at 365 nm to cleave the o-nitrobenzyl group, releasing the free fluorophore (Fig. 2.5). In one early demonstration of this approach, microtubule flux in the mitotic spindle was monitored following photoactivation of caged fluorescein-labeled tubulin (36). Similarly, a caged resorufin was used to observe intracellular actin filament dynamics (37). More recently, a series of cell permeable caged coumarin derivatives (38, 39) has been designed for the study of intercellular gap junctions. After intracellular delivery of these caged fluorophores, a small population of the caged coumarins was activated and used as a flu- orescent reporter to monitor the migration of the dye molecules through the gap junctions (39). The brightness of the fluorophore is a key consideration. When targeting cellular components that are expressed in low levels, the fluorescence signal from the optical reporter needs to
- 38. Labels and Probes for Live Cell Imaging 25 Fig. 2.5. Uncaging of non-fluorescent o-nitrobenzyl modified resorufin (top) and coumarin (bottom) derivatives after exposure to UV light. be bright. The brightness is defined as the product of the flu- orescence quantum yield and extinction coefficient of the fluo- rophore. For imaging low concentrations of cellular targets, weak fluorophores such as those based on NBD or pyrene may not be suitable. Some of the brightest fluorophores emitting in the visi- ble are based on rhodamine or BODIPY scaffolds. Both of these fluorophore classes are typified by quantum yields approaching unity and extinction coefficients of 80,000 M–1 cm–1 or more. In the far-red/NIR there are many bright fluorophores (6). Com- mon NIR-emitting cyanine dyes are typified by large extinc- tion coefficients often exceeding 200,000 M–1 cm–1 and quantum yields of 20% or greater (10). However, the fluorescence quantum yields of fluorophores with emission 800 nm begin to drop off considerably (34). This is the result of the relatively small energy difference between the ground and the excited states of these dyes, which allows for enhanced non-radiative decay of the flu- orophore from the excited state. The polarity of the fluorophore is an important factor in imaging agent design and may have a significant impact on the function of the fluorescent label. Many popular fluorescent labels are highly water-soluble polar species. Examples include AF488, fluorescein, sulforhodamine 101, and most cyanine-based far- red/NIR fluorophores. Imaging agents using polar fluorophores may not be able to cross the cell membrane by passive diffusion processes. Unless a targeted energy dependent transport mecha- nism is utilized, they are better suited for use as components of fluorescent reporters for imaging cell membrane or extracellular matrix components. Other fluorescent labels, such as DNA stains, rely on the permeability properties of the cell membrane for their function. These charged fluorescent molecules are often unable to penetrate healthy cells with intact membranes. If the membrane is compromised, as occurs with apoptotic or necrotic cells, these
- 39. 26 Hilderbrand dyes are able to enter the cell. One common method for preparing cell-permeable labels relies on the activity of intracellular esterases. The acetate or acetoxymethyl ester derivatives of many xanthene dye derivatives, such as fluorescein, are non-fluorescent, and non- polar, so that they may enter the cell via passive diffusion pro- cesses. Once inside the cell, the fluorescence signal of these fluo- rophores may be unmasked by intracellular esterase activity, which cleaves the acetyl groups from the fluorescein backbone, regener- ating fluorescein. The free fluorescein is negatively charged under physiological conditions and therefore becomes trapped inside the cell (Fig. 2.6). Fig. 2.6. Internalization of non-polar fluorescein diacetate followed by cleavage of the acetate groups by intracellular esterases, releasing polar fluorescein, which is trapped inside the cell. Many of the more elaborate commercially available fluo- rophores are expensive, often costing over $200/mg. Certain imaging applications may require large quantities of probe, espe- cially those involving in vivo microscopy. There are several more affordable fluorophore options with fluorescence emission in the visible range, such as fluorescein and rhodamine isothiocyanate derivatives (7). In contrast, there are few inexpensive commer- cially available NIR-emitting fluorophores, although efficient and inexpensive routes to prepare conjugatable fluorophores emitting in the NIR from commercially available precursors have been developed. The most common synthetic method is via nucle- ophilic attack on chloride containing carbocyanine precursors to install carboxylic acid functionality (34, 40, 41). These reac- tions can often be performed in a simple one-pot procedure with 90% efficiency and do not require any purification step (34, 42). 2.2. Quantum Dot Labels Luminescent semiconducting nanocrystals (QDs) are commonly used as labels for imaging at the cellular and subcellular levels (43 , 44). As with small molecule fluorophores, QDs have been
- 40. Labels and Probes for Live Cell Imaging 27 used for imaging a variety of cellular and subcellular targets. For example, targeting HER2 receptors on breast cancer cells and cytoplasmic actin and microtubule fibers has been demonstrated (44). Quantum dots are available with amine or carboxylic acid surface groups for bioconjugation reactions and come in a wide range of emission colors from the visible to NIR (6). Unlike organic dyes, quantum dots may be excited over a broad wave- length range with the highest extinction coefficients, often greater than 1,000,000 M–1 cm–1 , observed in the UV. These materials have several advantages over traditional fluorophores. The broad excitation range of QDs allows for simultaneous excitation of multiple quantum dots with different emission wavelengths using a single wavelength light source. Furthermore, QDs are not susceptible to rapid photobleaching under intense excitation, and therefore may be more suitable for confocal and other microscopy techniques, which require prolonged high intensity light exposure. Despite their significant advantages, QDs are not ideal for all imaging applications. Issues concerning QD blinking may complicate single molecule imaging experiments. Many quantum dots materials contain toxic cadmium (45), which was recently shown to leach out of the nanocrystal cores into the surrounding environment under certain biologically rele- vant conditions (46). In addition, commercially available QDs typically have a hydrodynamic diameter of 20–30 nm, signifi- cantly larger than small molecule organic fluorophores. The large size of the quantum dots may be a liability for imaging appli- cations where the size of the fluorescent reporter could inter- fere with the function of the biological process under inves- tigation. Actin fibers labeled with QDs have a proportionally decreased percent motility when compared to the correspond- ing AF488 organic fluorophore labeled filaments (47). Addition- ally, larger QDs may not be suited for monitoring fast diffus- ing neurotransmitters (48). As a result of their potential limita- tions for monitoring certain cellular processes, significant effort has been put forth to design improved quantum dots for live cell imaging applications. A large fraction of the typical QD diameter comes from polymer surface coating of the particles; therefore, efforts to decrease the thickness of this coating while maintaining ideal solubility characteristics could open QDs to new potential imaging applications. Following this strategy, QDs employing a short polyethylene glycol modified dihydrolipoic acid head group with a hydrodynamic diameter of 11 nm have been reported (49). Furthermore, the new smaller QDs have been engineered to contain only one site for biological labeling (49). Glutamate receptors labeled with the new, smaller QDs displayed a demonstrably improved ability to diffuse into neu- ronal synapses in comparison the corresponding commercial QD labeled receptors.
- 41. 28 Hilderbrand 2.3. Genetically Encoded Labels The researchers Roger Tsien, Martin Chalfie, and Samu Shimo- mura were recently awarded the 2008 Nobel prize in chemistry for their pioneering research on the identification, cloning, and modification of fluorescent proteins (50). Like QDs, genetically encoded fluorescent or chemiluminescent proteins are becoming commonplace. Recent reviews provide a comprehensive overview (51, 52). As with quantum dots and organic fluorophores, attention has been paid to developing fluorescent proteins in a rainbow of emission colors. Dozens of variants of these fluores- cent proteins have been detailed in the literature (51), several of which are in use today with emission in the blue, green, yellow, orange, red, and far red from EGFP, EYFP, mOrange, mCherry, and mPlum, respectively. Unlike QDs and small molecule fluo- rophores, these species are useful in imaging applications where they can be used to monitor gene expression (53). Fluorescent proteins are also well suited for investigation of chemotaxis. Flu- orescent protein expressing cells were used to investigate the role of the hematopoietic protein-1 (HEM-1) complex in cell motility (54, 55). The use of fluorescent proteins has been advantageous for the investigation of cell mitosis after challenge of human MDA cells with the anti-mitotic chemotherapeutics docetaxel (56) and paclitaxel (57). Bioluminescent enzymes, like fluorescent proteins, are geneti- cally encoded labels, although they require an additional substrate to generate a luminescent signal. Bioluminescent proteins have been isolated from a variety of organisms such as Photinus pyralis (firefly) (58), Renilla reniformis (sea pansy) (59), and Pyrophorus plagiophthalamus (click beetle) (60,61) with emission at ∼480, ∼560, and ∼600 nm, respectively. The firefly and click beetle luciferases use luciferin, whereas the sea pansy luciferase requires colenterazine as a substrate. The lux operon may be used to inves- tigate bacterial systems. This operon encodes both the luciferase and other proteins necessary for synthesis of the luciferin substrate (62, 63). 3. Responsive Probes The use of targeted fluorescent labels and genetically encoded flu- orophores has been invaluable in expanding our understanding of how the molecular machinery of the cell functions. However, these probes do not provide a detailed direct view of the function of many signaling molecules and messengers involved in cellular function. To investigate the interactions of these molecules, acti- vatable or switchable smart probes are necessary.
- 42. Labels and Probes for Live Cell Imaging 29 The phenomenon of fluorescence is a particularly versa- tile process, with many different parameters that can be uti- lized for development of activatable probes. These properties include fluorescence intensity shifts, wavelength shifts, chemilu- minescence activation, and fluorescence lifetime changes. Of these photophysical properties, most often biochemical probes are based on strategies to develop turn-on or wavelength-shift probes. Optimized fluorogenic probes share many selection cri- teria with targeted fluorescent labels. Factors to consider include biocompatibility and water solubility of the probes, suitability for extracellular or intracellular delivery, brightness of the fluo- rophore, and fluorescence excitation and emission wavelengths. In addition to these variables, other circumstances may influence the choice of probe. For example, selectivity of the imaging agent for the enzyme or analyte of interest is an important considera- tion. It is typically quite difficult to design a fluorogenic probe that displays complete selectivity to the target of interest. Vir- tually every known probe displays at least some basal activation by competing analytes or enzymes. The magnitude and mode of the fluorescence response is another factor. Typically activatable probes displaying an increase in emission or shifts in the absorp- tion or emission spectra are preferred. Many turn-off fluorescence based sensors have been designed, but these agents are more dif- ficult to use for cell imaging due to complications arising from detecting fluorescence decreases by microscopy. Turn-on probes can often be designed to show extremely strong fluorescence acti- vation, often over 100-fold, but may not be suitable for exper- iments where quantitative measurements are required. With an off–on fluorescence response, it is difficult to account for base- line fluorescence signal arising from the non-activated probe and variations in the local concentration of the imaging agent. When quantitative measurements are required, sensors with a ratiomet- ric fluorescence response are preferred. These sensors are suitable for quantitative measurements of analyte concentration because they allow for determination of the fluorescence activation in a manner independent of the local probe concentration. In the fol- lowing sections, an overview of current turn-on and wavelength- shift fluorescence-based probes for bioimaging will be presented. 3.1. Enzyme Activation Many enzyme activatable probes are based on the well-known phenomenon of self-quenching by organic fluorophores when held in close proximity to each other. An early example of this strategy in a fluorogenic probe suitable for use with live cells is a NIR-emitting fluorescent sensor for cathepsin D activity (Fig. 2.7). Cathepsin D is an aspartic protease that is known to be over- expressed in breast cancer cells. The probe consists of a polylysine polymer backbone modified with polyethylene glycol (PEG) poly- mers on the lysine side chains to improve solubility of the probe.
- 43. 30 Hilderbrand Fig. 2.7. Enzymatic activation of a polylysine based NIR activatable probe for cathepsin D. In addition, several of the lysine side chains are further modified with a cathepsin D specific cleavage sequence containing NIR- emitting Cy5.5 fluorophores (64). Up to 24 Cy5.5 fluorophores are incorporated per polylysine polymer. The high density of fluorophores allows for efficient self-quenching of the dyes. In this case, more than 99% of the fluorescence emission of the Cy5.5 flu- orophores is quenched in the probe (64). Upon cleavage of the probe with cathepsin D, up to 60-fold increase in fluorescence sig- nal is possible. The effectiveness of this probe was demonstrated in vivo by imaging of mice bearing cathepsin D positive tumors where a signal-to-noise ratio of up to 22.8 was reported between tumor and non-target tissue (65). This flexible design strategy for enzyme activatable probes is useful for both endo- and exopep- tidase enzymes. In addition to cathepsin D, fluorogenic probes for other enzymes such as cathepsin K, caspase-1, and MMP-2 utilizing this activation strategy have been reported (66–68). An alternative strategy employed for the design of enzyme activatable probes does not rely on dye–dye quenching interac- tions. Instead, its fluorescence switching is based on chemical modification of the fluorophore reporter to alter its optical emis- sion properties. One method for achieving this is by alterating the electronic structure of the fluorophore via formation of covalent bonds on portions of the fluorophore that are directly involved in fluorescence emission. For example, many classes of fluorophores such as 7-amino coumarins, rhodamines, fluoresceins, and Nile blue derivatives have amine or phenoxy groups that are part of their conjugated chromophore system and are available for chem- ical modification. Modification of these groups often has a dra- matic effect on the fluorescence emission of the fluorophore. These changes are typified by strong hipsochromic shifts of the absorption maximum of the dye and a concomitant blue shift of
- 44. Labels and Probes for Live Cell Imaging 31 the emission maximum. In many cases, the quantum yield of the modified fluorophore is also significantly decreased. Enzyme acti- vatable probes using this strategy can often show greater than 100-fold activation. This approach is useful for detection of enzy- matic activity from exopeptidases. Many of these fluorogenic enzyme substrates have been prepared by conjugation of an amine group on 7-amino coumarin or rhodamine 110 to the carboxy terminus of a peptide sequence specific for the enzyme of inter- est. Formation of the amide bond on the coumarin or rhodamine abolishes the characteristic fluorescence emission at approximately 430 or 520 nm for the coumarin and rhodamine, respectively. Enzyme action on the substrate releases the coumarin and restores its fluorescent signal. A range of fluorogenic probes for peptidases activated by cathepsins (69), caspases (70, 71), elastases (72), and trypsin (72) have been developed using this approach. This strat- egy has also been adapted to fluorogenic probes for sugars and phosphatases (73). 3.2. Metal Ion Sensing The design of effective probes for metal ions faces many chal- lenges. The primary concerns are selectivity, metal ion affinity, and fluorescence response. There are numerous activatable and ratio- metric fluorescence-based sensors for detection of bio-relevant ions such as Ca2+ , Mg2+ , Na2+ , K2+ , Zn2+ , Cu2+ , Fe3+ , and H+ . However, with the exception of pH responsive sensors (H+ ions), nearly every metal ion probe has side reactivity with analytes other than the targeted metal ion. Therefore the presence or absence of potential interfering ions influences the probe choice. The affin- ity of the analyte to the probe is another factor. The Kd values for analyte dissociation from the fluorescence-based sensor should be matched to the expected concentration of the ion under investi- gation to yield optimal response. Fluorescent probes for calcium ions form one of the most diverse classes of metal ion sensors. The wide array of probes is in part due to the importance of calcium as a signaling molecule in biology. Cellular Ca2+ plays many functional and regulatory roles from muscle fiber contraction to signal transduction. Dozens of Ca2+ responsive sensors have been detailed in the literature, and a complete review of these probes is beyond the scope of this chapter. For more detailed information on Ca2+ probes, there are several excellent literature reviews (74, 75). Calcium ion probes can be divided into two broad categories: intensity based and ratiometric. Probes in both classes have a wide range of reported Kd values. For example, the fluorogenic Oregon Green 488 BAPTA-1, -6F, and -5 N probes, which are all based on the BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid) chelating group have tunable Ca2+ Kd values. By varying the substituents on the BAPTA chelator, the Kd for Ca2+ can be altered from 170 nM to 20 M (Fig. 2.8) (6). Many low affinity
- 45. 32 Hilderbrand Fig. 2.8. Fluorogenic oregon green-based Ca2+ selective probes. The K d values of these sensors can be tuned by altering the electron-withdrawing properties of the substituents on the BAPTA chelating moieties. calcium binders also display selectivity for Mg2+ . The Mg2+ probe Mag-Fluo-4 has Kd values of 4.7 mM and 22 M for Mg2+ and Ca2+ , respectively (6). The Mg2+ binding of this probe is well matched to the typical sub- to low-millimolar cellular magne- sium levels (76). Ratiometric probes for Ca2+ can either show a change in absorption wavelength or emission wavelength upon coordination of the divalent ion. Examples include Fura-2 and Indo-1 for absorption and emission wavelength shift sensors, respectively (77). Ratiometric response with both classes of probes is possible, although sensors showing shifts in fluorescence emission are preferable since only one excitation source is nec- essary. This is particularly important in applications where laser excitation is used or where analysis will be performed by flow cytometry. In addition to activatable probes for calcium, which were first reported in the early 1980s (78), recent years have seen the devel- opment of selective probes for many other metal ions. In the past decade significant attention has been directed toward imaging intracellular zinc to investigate its roles in biological homeosta- sis and signal transduction. Here we give an overview of several widely applied sensors for zinc. For a more complete survey of current Zn2+ selective probes, please see one of several recent reviews (79–81). Much of the pioneering work on design of effi- cient fluorescence-based zinc sensors originated in the Lippard and Nagano laboratories. The Lippard lab has reported a series of fluorescein-based probes for zinc using the dipicolylamine chelat- ing group (82–85). The earliest of these probes, Zinpyr-1 displays a threefold increase in emission upon binding Zn2+ and has a 0.7 nM binding affinity (82). Sensors with decreased Zn2+ affinity have subsequently been prepared via replacement of one of the pyridyl arms of the dipicolylamine chelating motif with thiophene or thioether coordinating groups, decreasing the binding affinity
- 46. Labels and Probes for Live Cell Imaging 33 Fig. 2.9. Representative Zn2+ selective sensors. ZP2 and ZS5 are intensity based turn-on probes with sub nanomolar and micromolar Zn2+ affinities, respectively. ZNP1 is a ratiometric sensor with dual emission at 545 and 624 nm. from the low nM to M (Fig. 2.9) (86). One of these low-affinity probes, ZS5 was used to visualize glutamate-mediated Zn(II) uptake in dendrites and Zn(II) release resulting from nitrosative stress (86). As with Lippard, Nagano has focused on design of flu- orescent Zn2+ sensors based on the fluorescein scaffold. Probes of the ZnAF family have low fluorescence background and strong activation of up to 69-fold upon Zn2+ coordination (87, 88). These sensors have been used to visualize Zn2+ release in the rat hippocampus (88) and to monitor presynaptic Zn2+ pools (89). Systematic modification of the dipicolylamine chelating moiety on these probes has enabled preparation of ZnAF probes with Kd val- ues ranging from 2.7 nM to 600 M (90). The ratiometric Zn2+ probes FuraZin and IndoZin, which are based on the related Fura and Indo Ca2+ sensors, (91) were used to monitor intracellular zinc uptake (92). Both FuraZin and IndoZin are excited at short wavelength (400 nm) (91). To minimize potential phototoxic effects of UV excitation, long-wavelength ratiometric probes such as Zin-naphthopyr-1 (ZNP1), which has a 0.55 nM Zn2+ affin- ity, were designed (Fig. 2.9) (93). The ZNP1 probe has dual emission at 545 and 624 nm, where increasing [Zn2+ ] induces a dramatic increase in the 624-nm emission signal. The diacetate derivative of this probe is membrane permeable and was used to image release of Zn2+ from COS-7 cells in real time (93). A NIR- emitting ratiometric probe, DIPCY, is based on a carbocyanine fluorophore scaffold (94) This probe, which has a Zn2+ Kd of 98 nM, displays an approximate 50 nm red-shift in its absorbance spectrum upon binding zinc. Ion selective probes for H+ are one of the oldest and most studied classes of ion sensors. Their development and use has been vital for investigation of pH changes in the endosomal/lysosomal system. Furthermore, disruption of acid/base homeostasis is asso- ciated with the pathophysiology of diseases such as cancer, cystic fibrosis, and immune dysfunction (95–98). Many fluorophores
- 47. 34 Hilderbrand have intrinsic pH sensitivity. For example, fluorescein has a flu- orogenic pKa of approximately 6.4 and has been used as a dual excitation single emission ratiometric probe for intracellu- lar pH (99, 100). However, fluorescein can leak from cells and is used infrequently as a stand-alone probe for intracellular ratio- metric pH imaging. New nanomaterials doped with FITC have been developed for ratiometric pH imaging. In one example, fluorescein and rhodamine isothiocyanate fluorophores (the rho- damine is used as a pH insensitive reference) were incorpo- rated into core/shell silica nanoparticles and used for monitoring pH in intracellular compartments of mast cells (101). The dual excitation-single emission pH probe BCECF is one of the most widely used pH probes and is a fluorescein derivative modified with two carboxyethyl groups in the 2 and 7 positions of the dye. These additional carboxylate groups significantly improve intra- cellular retention of the sensor and contribute to an increase in the pH responsive pKa to 6.97 (102). However, as a result of its dual excitation single-emission response, it is not ideal for imag- ing with laser microscopes or for flow cytometry experiments. To address this, single excitation dual emission pH responsive fluorophores were developed. In the early 1990s the seminaph- thorhodafluor scaffold was designed for ratiometric pH imag- ing (103). In addition to having a single excitation dual emis- sion response to pH with a pKa of approximately 7.5, the probe exhibits red-shifted emission between 600 and 640 nm (103). One of these derivatives, carboxy-SNARF-1 has been used for imaging intracellular pH in chicken embryo epithelial cells (104). Probes for sensing pH can also be combined with targeting strate- gies. A series of pH responsive fluorogenic boron-dipyrromethene (BODIPY) fluorophores with tunable pKa values between 3.8 and 6.0 were recently reported. These fluorophores can be conjugated to targeting groups such as trastuzumab for use as fluorescence- based switches and are activated by internalization into the endo- somal/lysosomal system of cancer cells (105). NIR fluorescent probes for pH sensing show potential for use in intracellular and in vivo pH measurement. Most current NIR pH probes are based on the carbocyanine scaffold. In one approach, dealkylation of one or both of the indole nitrogens on a non-pH responsive carbocyanine fluorophore renders it sensi- tive to pH (Fig. 2.10) (106–109). In contrast to xanthene based pH sensors, the carbocyanine dyes show an increase in fluores- cence emission as the pH decreases. These probes have been used to monitor agonist-induced G protein-coupled receptor internal- ization into CHO or Hek293 cells (106). One pH responsive dye HCyC-646 with a fluorogenic pKa of 6.2 and fluorescence emis- sion at 670 nm was paired with pH insensitive Cy7 fluorophores on a bacteriophage particle scaffold for use as an nanoscale NIR ratiometric pH sensor (109). This system was used to monitor
- 48. Labels and Probes for Live Cell Imaging 35 Fig. 2.10. The pH-dependent equilibrium showing activation of HCyC-646 at acidic pH (panel A). Absorption traces for HCyC-646 at pH 8 and pH 4 and fluorescence emission at pH 4, dashed, solid, and dotted lines, respectively (panel B). intracellular pH following internalization into RAW cells and its potential for imaging pH in small animal models was demon- strated (109). Although much recent work has focused on development of ion-selective probes specific for Ca2+ , Zn2+ , and H+ , many probes for other bio-relevant analytes have been developed. Ratiometric fluorescent indicators based different-sized crown ether ion chelators have been reported for Na+ (SBFI, Kd = 3.8 mM) (110) and K+ (PBFI, Kd = 5.1 mM) (110, 111), although they have relatively poor ion selectivity. Due to the potential role of unregulated cellular copper, in various diseases from amyotrophic lateral sclerosis (112) to Alzheimer’s disease (113), fluorogenic copper specific probes have been designed (114–116). Additional efforts have focused on preparation of iron selective probes, but only a few turn-on sensors have been reported (117–120) and their ability for live cell imaging remains relatively untested. 3.3. ROS Sensing There is significant interest in detection of reactive oxygen (ROS) and reactive nitrogen species (RNS) in biology. These reac- tive compounds are involved in multiple signal transduction and
- 49. 36 Hilderbrand regulatory processes. Furthermore, many of these compounds are strong oxidants and play critical roles in host defense and, when unregulated, in disease progression. One of the key challenges is the development of sensors with a high degree of specificity for a single analyte. This is often difficult since many of these species exhibit similar behavior as oxidants. Without selective sensors, it is very difficult to study the function of a single ROS/RNS since many different species are present simultaneously in the same biological systems. The inability to differentiate between specific reactive species has been the primary flaw of early probes such as 2 ,7 -dichlorodihydrofluorescein (DCFH), which shows broad non-specific activation to a variety of oxidant species (121). In addition to this lack of selectivity, DCFH displays marked autoxi- dation activity when exposed to light. Nitric oxide (NO) is one of the first reactive species for which selective fluorogenic sensors were developed. There are many approaches to imaging NO, and a more complete sum- mary is given elsewhere (122, 123). The most common strat- egy today for design of selective NO sensors is based on the o-phenylenediamine functional group. In the presence of dioxy- gen and NO, a selective reaction occurs to convert the o-diamine into a triazole derivative (Fig. 2.11a). This effectively results in an increase in fluorescence signal since the amine groups of the o-phenylenediamine group, which are good photoinduced electron transfer (PET) quenchers, are converted into an elec- tron deficient triazole. This approach has been used for design of a variety of NO sensors using naphthalene (124), fluores- cein (125), BODIPY (126), rhodamine (127), and carbocyanine (128) fluorophores spanning the electromagnetic spectrum from the blue to NIR. In general, these probes show excellent selectiv- ity for NO in aerobic environments with little or no observed reactivity to other oxidants such as peroxynitrite (ONOO– ), hydrogen peroxide (H2O2), or superoxide radical (O2 – ) (125). Although useful in most imaging applications, these sensors do not directly monitor NO. Fluorogenic sensors for NO based on the o-phenylenediamine functional group only react with RNS formed by the pre reaction of NO with O2. Therefore detection is dependent not only on the presence of NO but also local O2 levels. A preferred tactic is fluorogenic sensors that are capable of direct reaction with NO. Recently the first probes suitable for live cell imaging based on direct detection of NO were reported (129). These sensors consist of a Cu(II) complex with a modi- fied fluorescein derivative bearing an 8-aminoquinaldine chelat- ing group (130). The paramagnetic properties of the Cu(II) coordinated to the fluorescein probe result in quenched fluores- cence emission in the absence of NO. Reaction of this probe, CuFL, with NO results in reduction of the Cu(II) to Cu(I),
- 50. Other documents randomly have different content
- 51. every country gentleman; but in kind and in degree, the same character and spirit extend to all such life, and I have therefore taken the liberty of transcribing Mr. Willis’s sketch as completely as my limits would admit. Nothing, were a volume written on the subject, could bring it more palpably and correctly before the mind of the reader; and I think that if there be a perfection in human life, it is to be found, so far as all the goods of providence and the easy elegances of society can make it so, in the rural life of the English nobility and gentry.
- 52. CHAPTER IV. THE ROUTINE OF COUNTRY SPORTS. In my last chapter I took a view of the variety given to rural life by the annual visit to town: but if a gentleman have no desire so to vary his existence; if he love the country too well to leave it at all, most plentiful are the resources which offer themselves for pleasantly speeding on the time. If he be attached merely to field sports, not a moment of the whole year but he may fill up with his peculiar enjoyment. Racing, hunting, coursing, shooting, fishing, all offer themselves to his choice; and rural sports, as every thing else in English life, are so systematized; every thing belonging to them is so exactly regulated; all their necessary implements and accessories, are brought to such an admirable pitch of perfection by the advancement of the arts, that the pleasures of the sportsman are rendered complete, and are diffused over every portion of the year. Field sports have long ceased to be followed in that rude and promiscuous manner which they were when forests overrun the greater part of Europe, and hunting was almost necessary to existence. Parties of hunters no longer go out with dogs of various kinds—greyhounds, hounds, spaniels, and terriers, all in leash, as our ancestors frequently did, ready to slip them on any kind of game which might present itself, and with bows also ready to make more sure of their prey. We have no battues, such as are still to be found in some parts of the continent, and which used to be the common mode of hunting in the Highlands, when the beasts of a whole district were driven into a small space, and subjected to a promiscuous slaughter; a scene such as Taylor the water-poet describes himself as witnessing in the Braes of Mar; nor such as those perpetrated by the King of Naples in Austria, Bohemia, and Moravia, in which he killed 5 bears, 1820 boars, 1950 deer, 1145 does, 1625 roebucks, 1121 rabbits, 13 wolves, 17 badgers, 16,354 hares, 354 foxes, 15,350 pheasants, and 12,335 partridges. Such scenes are not to be witnessed in this country. Every field sport is
- 53. here become a science. Hunting, coursing, shooting, each has its own season, its well-defined bounds, its peculiar horses, dogs, and weapons. Our horses and dogs, by long and anxious attention to the preservation of their specific characters, and to the improvement of their breed, are become pre-eminent, each in their own department. Our sporting nobility and gentry have not contented themselves with becoming thoroughly skilful in every thing relating to field diversions; but have many of them communicated their knowledge through the press to their countrymen, and have thus furnished our libraries with more practical information of this kind than ever was possessed by any one country at any one time; and contributed to make these pursuits as effective, elegant, and attractive as possible. It is not my province to go into the details of any particular sports; for them I refer the reader to Daniel, Beckford, Col. Thornton, Sir John Sebright, Col. Hawker, Tom Oakleigh, Nimrod, and the sporting magazines. My business is to shew how gentlemen may and do spend their time in the country. And in the mere catalogue of out-of- door sports, are there not racing, hunting, coursing, shooting, angling? Hawking once was an elegant addition to this list; but that has nearly fallen into disuse in this country, and may be said to exist only in the practice of Sir John Sebright, and the grand falconer of England, the Duke of St. Albans. Archery too, once the great boast of our forests, and the constant attendant on the hunt, has, as a field exercise, followed hawking. It has of late years been revived and practised by the gentry as a graceful amusement, and an occasion for assembling together at certain periods in the country; but as an adjunct of the field sports it is past for ever. Racing, every one knows, is a matter of intense interest with a great portion of the nobility, gentry, and others; and those who delight in it, know where to find Newmarket, Epsom, Ascot Heath, Doncaster, and other places, often to their cost: almost every county and considerable town, has its course and annual races. These, however, to the country gentleman, unless he be one whose great and costly passion is for breeding and betting on race-horses, are but occasional excitements: the rest run their round of seasons as regularly as the seasons themselves; and place a lover of field sports in the country
- 54. at any point of the year, and one or more of them are ready for his enjoyment. Is it winter? He has choice of all, except it be angling. Hunting, coursing, shooting, are all in their full season. Hunting, as I have said, is more confined in its range than it was anciently; but it is more regular, less fatiguing, less savage in its character, more complete in its practice and appointments. There is now neither the boar, the bear, nor the wolf, to try the courage of our youth, and stag and buck hunting may be considered as rare and almost local amusements,—but we may quote the words of a great authority as to the position which hunting occupies amongst the rural sports of England. “There is certainly no country in the world, where the sport of hunting on horseback is carried to such a height as in Great Britain at the present day, and where the pleasures of a fox-chase are so well understood, and conducted on such purely scientific principles. It is considered the beau idéal of hunting by those who pursue it. There can be no doubt, that it is infinitely superior to stag- hunting, for the real sportsman can only enjoy that chase, when the deer is sought for, and found like other game which are pursued by hounds. In the case of finding an out-lying fallow-deer, which is unharboured in this manner, great sport is frequently afforded; but this is rarely to be met with in Great Britain: so that fox-hunting is now the chief amusement of the true British sportsman: and a noble one it is—the artifices and dexterity employed by this lively, crafty animal, to avoid the dogs, are worthy of our admiration, as he exhibits more devices for self-preservation than any other beast of the chase. In many parts of this and the sister island, hare-hunting is much followed, but fox-hunters consider it as a sport only fit for women and old men,—but, although it is less arduous than that of the fox-chase, there are charms attached to it which compensate for the hard riding of the other.” I do not enter here into the question of cruelty in this sport, nor into the other question of injury resulting from it to crops and fences, on which grounds many so strongly object to hunting, and on the former ground, indeed, to all field sports. Lord Byron, for instance, thought hunting a barbarous amusement, fit only for a barbarous country. It is not my intention to undertake the defence of
- 55. this old English sport from the standing charge against it, we here have only to deal with it as a feature of rural life; and though one cannot say much in praise of its humanity, it cannot be denied that it is a pursuit of a vigorous and exciting character. A fine field of hunters in their scarlet coats, rushing over forest, heath, fence or stream, on noble steeds, and with a pack of beautiful dogs in full cry, is a very picturesque and animating spectacle. Through the winter, then, up to the very approach of spring, hunting offers whatever charms it possesses; pheasant, woodcock and snipe shooting, in the woods and by the streams, are in all their glory. It is the time for pursuing all manner of wild fowl, in fens and along the sea-coast; and if any one would know what are the eager and adventurous pleasures of that pursuit, let him join some old fowler for a week amongst the reeds of Cambridge, Huntingdon, or Lincolnshire,—now laying his traps and springes, now crouching amongst the green masses of flags and other water plants, or crawling on hands and knees for a shot at teal, widgeon, or wild duck; now visiting the decoys, or shooting right and left amongst the rising and contorting snipes. Or let him read Col. Hawker’s delightful description of swivel shooting on the coasts, the mud-launchers and followers of the sea flocks by night. Those are sports which require a spice of enthusiasm and love of adventure far above the pitch of the ordinary sportsman. When spring arrives, and warns the shooter to give rest to the creatures of his pursuit, that they may pair, produce, and rear their broods; as he lays down the gun, he can take up the angle. Many a keen and devoted old sportsman, however, never knows when to lay down the gun. Though he will no longer fire at game, he likes through the spring and summer months to carry his gun on his arm through the woods, to knock down what he calls vermin,—stoats, weazels, polecats, jays, magpies, hawks, owls; all those creatures that destroy game, or their young broods, or suck their eggs. He is fond of spying out the nests of partridges and pheasants, and from time to time marking their progress. It is a grand anticipative pleasure to him when, passing along the furrow of the standing corn, his old pointer, or favourite spaniel starts the young birds just
- 56. able to take the wing, and he counts them over with a silent exultation. He is fond of seeing to the training of his young dogs, of selecting fresh ones, of putting his fowling-pieces and all his shooting gear in order. There are some old sportsmen of my acquaintance, who, during what they call this idle time, have made collections of curious birds and small animals which might furnish some facts to natural history. An old uncle of mine in Derbyshire, who has shot away a fine estate, I scarcely ever recollect to have seen out of doors without his gun. I saw him lately, when in that county, a feeble, worn-out old man, just able to totter about, but still with the gun on his arm. For those, however, who can find it in their hearts to lay aside the gun at the prescribed time, and yet long for rural sports, what can so delightfully fill up the spring and summer as the fishing-rod? There is no rural art, except that of shooting, for which modern science and invention have done so much as angling. Since Izaak Walton gave such an impetus to this taste by his delicious old book, it has gradually assumed a new and fascinating character. A host of contrivances have been expended on fishing tackle. What splendid rods for simple angling, trolling, or fly-fishing, are now offered to the admiring eyes of the amateur! what a multitude of apparatus of one kind or other! what silver fish and endless artificial flies Angling has become widened and exalted in its sphere with the general expansion of knowledge and the improvement of taste. It has associated itself with the pleasures and refinements of literature and poetry. All those charms which worthy Izaak threw round it, have continued to cling to it, and others have grown up around them. The love of nature, the love of travel have intertwined themselves with the love of angling. Angling has thence become, as it were, a new and more attractive pursuit—a matter of taste and science as well as of health and pleasure. It is found that it may not only be followed by the tourist without diverting him from his primal objects, but that it adds most essentially to the delights of a summer excursion. Since Wordsworth and John Wilson set up their “Angler’s Tent” on the banks of Wast-Water, “at the head of that wild and solitary lake, which they had reached by the mountain-path that passes Barn-Moor-Tarn from Eskdale,” making an angling excursion
- 57. of seven days amongst the mountains of Westmoreland, Lancashire, and Cumberland, having “their tent, large panniers filled with its furniture, provisions, etc., loaded upon horses, which, while the anglers, who separated every morning, pursued each his own sport up the torrents, were carried over the mountains to the appointed place, by some lake or stream, where they were to meet again in the evening;” and that solitary trade, Mid rural peace in peacefulness pursued, Through rocky glen, wild moor, and hanging wood, White flowering meadow, and romantic glade; since Sir Humphry Davy went angling and philosophising in the mountain tarns, and along the trout and salmon streams not only of Scotland and Ireland, but of France and Switzerland, the enthusiasm for angling has grown into a grand and expansive passion. We have our “Anglers in Wales,” our “Anglers in Ireland;” Stephen Oliver has flourished his lines over the streams of the north, Jesse over the gentle and majestic Thames. The only wonder is, that, as our countrymen walk to and fro through all known regions of the earth, we do not hear of anglers in the Danube—the Ister—the Indus—the Joliba,—of trolling in La Plata, and fly-fishing in South Africa and Australia. All that will come in its own good time: meanwhile let us remind our country friends of the further blessings which await them, even should all the rapid streams of our mountain rivers and rivulets, Loch Leven trout, Loch Fine herrings, and salmon pulled flouncing from the crystal waters of the Teith or the Shannon, to be crimped and grilled by most delicious art, satiate them before the summer is over. The 12th of August approaches! the gun is roused from its slumber—the dogs are howling in ecstasy on their release from the kennel—the heather is burst into all its crimson splendour on the moors and the mountains, and grouse-shooting is at hand once more! That sentence is enough to make a sportsman start to his feet if it were but whispered to him in his deepest after-dinner doze. In “The Book of the Seasons” I asserted that sportsmen felt the animating
- 58. influence of nature and its beauty in their pursuits. For that passage many have been the gentle lectures of the tender-hearted; but that it was a true passage has been shewn by the thanks which many sportsmen have given me for that simple vindication, and by the repeated quotation of the whole article in their books. That they do feel it, is plainly shewn in many papers of the sporting magazines; but nowhere more vividly than in “The Oakleigh Shooting Code.” If the unction with which the paper on grouse-shooting is written in that book were more diffused through works of the like nature, vain would be all arguments to check the love of shooting. The feeling on this subject has been evidenced by the avidity with which that part of the book has been quoted far and wide. But the spirit of the picturesque is not more prominent in these chapters than in the description of Oakleigh Hall, and of the “wide-ranging treeless view of the smooth-turfed limestone hills, the white rocks breaking out in patches, so characteristic of Derbyshire.” But we are pausing on our way to the Highlands; and surely nothing can be so inspiring and exciting in the whole circle of sporting scenes as a trip to the moors and mountains of the north, in the height of summer—in the beauty of summer weather, and in the full beauty of the scenery itself. If the season is fine—the roads are dry—the walks are dry—the bogs are become, many of them, passable, the heather is in full bloom, the fresh air of the mountains, or the waters in sailing thither, the rapid changes of scene, the novel aspects of life and nature in progressing onward, by the carriage, the railway, the steamer, with all their varying groups of tourists and pleasure-seekers, of men of business and men of idleness, are full of enjoyment. To the man from the rich monotonous Lowlands, from the large town, from the heart of the metropolis perhaps, from the weary yoke of business, public or private, of law, of college study, of parliament and committees, what can be more penetrating and delicious than the breathing of the fresh buoyant air, the pleasant flitting of the breeze, the dash of sunny waters, the aspect of mountains and moors in all their shadow and gloom, or in their brightness as they rise in their clear still beauty into the azure heavens, or bask broad and brown in the noon-sun? There go the
- 59. happy sportsmen; seated on the deck of some fast-sailing steamer, with human groups around them; they are fast approaching the “land of the mountain and the flood.” They already seem to tread the elastic turf, to smell the heather bloom, and the peat fire of the Highland hut; to climb the moory hill, to hear the thunder of the linn, or pace the pebbly shore of the birch-skirted lake. They have left dull scenes or dry studies behind, and a volume of Walter Scott’s novels is in their hands, living with all the character and traditions of the mountain-land before them. Well then, is it not a blessed circumstance that our poets and romancers have kindled the spirit of these things in the heart of our countrymen, that such places lie within our own island, and that science has so quickened our transit to them? Let us just note a few of the symptoms which shew us that this memorable 12th of August is at hand. In the market towns you see the country sportsman hastening along the streets, paying quick visits to his gunsmith, ammunition dealer, tailor, draper, etc. He is getting all his requisites together. His dogs are at his heels. Then you see him already invested in his jacket and straw hat, driving off in his gig, phaeton, or other carriage, with keeper or companion, and perhaps a couple of dogs stowed away with him. You see the keeper and the dog-cart on their way too. As you get northward these signs thicken. In large towns, as Manchester, Liverpool, Glasgow, Edinburgh, you see keeper-like looking men, with pointers and setters for sale tied up to some palisade, or lamp-post, at the corner of a street. But woe to those who have to purchase dogs under such circumstances. It is ten to one but they are grievously gulled; or if they should chance to stumble upon a tolerable dog, there is not time for that mutual knowledge to grow up which should exist between the sportsman and his companion of the field. He that sees beforehand his trip to the hills, should beforehand have all in readiness: he who on a summer ramble is smitten with a sudden desire of grouse-shooting, must however, do the best he can. When you pass into Scotland, the signals of the time grow more conspicuous. In the newspapers, you see everywhere advertisements of Highland tracts to be let as shooting-grounds. When you get into the Highlands themselves, you find in all the inns
- 60. maps of the neighbouring estates, divided into shooting-grounds for letting. It is very probable that the income derived from this source by the Highland proprietors frequently far exceeds the rental of the same estates for the grazing of sheep and cattle. The waters and the heaths seem to be the most profitable property of a great part of the Highlands. Almost every stream and loch is carefully preserved and let as a trout or salmon fishery, many of them for enormous sums; and so far is this carried, that sportsmen who are not inclined to pay eighty or a hundred pounds a-year for a shooting ground, complain that Ireland is the only country now for shooting in any degree of freedom. Sometimes several gentlemen join at a shooting ground; and it is a picturesque sight to see them, and their dogs and keepers, drawing towards their particular locations as the day approaches. On the 10th of August, 1836, we sailed up the Grand Caledonian Canal from Fort William to Inverness in the steam-packet with a large party of these gentlemen. Of their number, principally military men—
- 61. Captains, and colonels, and men at arms; some notion may be formed from the fact that we had on board upwards of seventy dogs, mostly beautiful setters; a perfect pyramid of gun-cases was piled on the deck, and dog-carts and keepers completed the scene. One of the singular features of English life at the present moment is the swarming of summer tourists in all interesting quarters. In these Highland regions the consequent effect is often truly ludicrous. Into one miserable village, or one poor solitary inn, pour, day after day, the summer through, from seventy to a hundred people. The impossibility of such a place accommodating such a company is the first thing which strikes every one. The moment, therefore, that the vessel touches the quay, out rushes the whole throng, and a race commences to the house or village to secure beds for the night. Such is the impetus of the rush that the first arrivers are frequently driven by the “pressure from without” up the stairs to the very roof. A scene of the most laughable confusion is exhibited. All are clamouring for beds; nobody can be heard or attended to; and generally all who can, burst into rooms which are not locked up, and take forcible possession. Such scenes, any one who has gone up this canal, or to the Western Isles must have seen,—at Oban, at Tobermory, and at Inverness, which last place boasts three inns, and where, on our arrival with a hundred fellow-passengers, we found three hundred others had just landed from a London steamer! Our sportsmen, however, who were well aware of the statistics of the north, had written beforehand, and secured bed-rooms at all the sleeping-places, which were duly locked up against their arrival, and they sate very composedly to witness the race of worse-informed mortals. On this occasion a very characteristic contrast was presented between the sportsmen and a number of students who were on board at the time. These students, many of whom spend the college recess in pedestrianizing through the Highlands, have a character almost as peculiar to themselves as the German Bürschen. In twos and threes, with their knapsacks on their backs, they may be seen
- 62. rambling on, wherever there is fine scenery or spots of note to be visited. They step on board a packet at one place, and go off at another, steering away into the hills; ready to take up their quarters at such abode as may offer—the road-side inn or the smoky hut of the Gael. Wherever you see them, they are all curiosity and enthusiasm; all on fire with the sublime and beautiful—athirst for knowledge; historical, antiquarian, traditionary, botanical, geological —anything in the shape of knowledge. They are the first to climb the hill, to reach the waterfall, to crowd round every spot of tragic interest; everywhere they go agog with imagination, and everywhere they lament that they do not feel adequately, the power, and beauty, and grandeur of the objects of their attention. Such a group we had on board. On the other hand, the sportsmen had but one object, which absorbed all their interests and faculties. They cared not at that moment for the Fall of Foyers, saw scarcely the splendid mountains and glens around. Their souls were in the brown hills of their shooting grounds—the fever of the 12th of August was upon them. They kept together, talking of guns, dogs, grouse, roebucks; all their conversation was larded and illustrated with the phraseology of their own favourite pursuit. They were, many of them, clad in a close jacket and trousers of shepherd’s tartan, with their telescope slung at their backs. They seemed to look on the students as so many hair-brained and romantic striplings—the students on them, as so many creatures of the chase. As we proceeded, the fiery Nimrods were, one after another, put out at the opening of beautiful glens, and at the foot of wild mountains where their huts lay, and the vessel received a considerable accession of silence by the departure of their keepers, who, having found a Highland piper on board, got up a dance in the steerage cabin, and kept that end of the vessel pretty well alive both day and night. Having thus brought them to their grounds, there can be no better narrator of what passes there than Thomas Oakleigh. “On the 11th of August the sportsman arrives at his shooting quarters; probably some isolated tavern, ‘old as the hills,’—if such a house as the grouse-shooter occasionally locates himself in, in the northern or midland counties of England, or in Scotland, where
- 63. oatcake and peat supply the place of bread and fuel, can be called a tavern. The place, humble in character, has been the immemorial resort of sportsmen in August, although during the rest of the year, sometimes many months elapse ere a customer, save some itinerant salesman calling for his mug of beer, ‘darkens the door.’ * * * At the house will be found all the keepers, and tenters, and poachers, and young men from the country round, assembled, amounting in the whole to not more than some eight or ten persons, all knowing ones, each anxious to display his knowledge of the number and locality of the broods, but each differing, wide as the poles asunder, in his statement, except on four points, in which all are agreed, viz. —That the hatching season has been finer than was ever known before! That the broods are larger and more numerous than were ever counted before! That the birds are heavier and stronger than were ever seen before! and that they will, on the following day, lie better than they ever did on any previous opening day in the recollection of the oldest person present! Each successive season being, in their idea, more propitious than its precursor! Anxiety and expectation are now arrived at a climax. At night, the blithe and jocund peasantry mingle with their superiors: their pursuits are for once something akin. In the field-sports they can sympathize together: the peasant and the peer associate; the plough-boy and the squire talk familiarly together; it is the privilege of the former, his prescriptive right. The circling cup, and lighthearted and hilarious laugh promiscuously go round! This night distinctions are unknown— and would that it were oftener so! * * * Long before midnight, all who can obtain beds retire, though not an eye is drowsy. The retainers lie on sofas, elbow-chairs, or whatever else presents itself; but sleep is almost a stranger during the night. The soldier before battle, is not more anxious as to the result of the morrow, than is the sportsman on the night of the 11th of August! Morning dawns, ‘and heavily with mists comes on the day.’ The occupiers of benches and chairs are first on the alert: the landlady is called; breakfast is prepared—the dogs are looked at; all is tumult, noise, and confusion. Reckless must he be that can rest longer in bed—‘the cootie moor-cocks crowsely crow;’ breakfast is hastily dispatched—
- 64. next is heard the howling and yelping of dogs, the cracking of whips, the snapping of locks, the charging, and flashing, and firing of guns, and every other note of preparation. The march is sounded, and away they wind for the heather and hills, true peep-o’-day boys, far, far from the busy, money-getting world, to breathe empyreal air; to enjoy a sport that should be monopolized by princes—if, indeed, princes could be found deserving of such a monopoly! Every person the shooter meets with seems this day to have thrown off his sordid cloak, and to be divested of those meaner passions which render life miserable: all are now warm, open-hearted, frank, sincere, and obliging. The sportsman’s shooting-dress is a sibboleth, which introduces him alike to his superiors, to his fellows, and his inferiors: an acquaintance is formed at first sight: there are no distant looks, no coldness, no outpouring of arrogance, or avarice, or pride; but a happy rivalry exists, to eclipse each other in the number and size of birds killed—the chief object of emulation being to kill the finest old cock. Let us be understood to express that this happy state of things subsists only so long as the shooter’s peregrinations are circumscribed by the limits of his own or friend’s manor. The moment he becomes a borderer, a very different reception awaits him! To the sportsman in training, full of health and strength, and well appointed, it is of little consequence whether there be game or not. The inspiriting character of the sport, and the wild beauty of the scenery, so different from what he is elsewhere in the habit of contemplating, hold out a charm that dispels fatigue! He feels not the drudgery. To him the hills are lovely in every aspect; whether beneath a hot, autumnal sun, with not a cloud to intercept the torrid beam, or beneath the dark canopy of thunder-clouds; whether in the frosty morn or in the dewy eve—whether, when through the clear atmosphere he surveys, as it were in a map, the countries that lie stretched around and beneath him, or when he wanders darkly on, amidst eternal mists that roll continuously past him—still a charm pervades the hills. The sun shines brighter, and the storm rages more furiously than in the valleys! The very sterility pleases: and to him who has been brought thither by the rapid means of travelling now adopted, from some bustling mart of trade or vortex of fashion,
- 65. the novelty of loneliness is agreeably exciting! The stillness that reigns around is as wonderful to him as the solidity of land to the stranded sailor! Scarcely is there a change of scene—stillness and solitude, hill and ravine, sky and heather, everywhere magnificent, the outline everywhere bold, and where the view terminates amid rocks and crags, frequently sublime! At noonday, near some rocky summit, perchance on the shepherd’s stone, the shooter seats himself, and shares his last sandwich with his panting dogs. We will suppose him to be on the boundary of the muir-lands: on one hand he sees an unbounded expanse of heathery hills, by no means monotonous if he will look upon them with the eye of a painter, for there is every shade of yellow, green, brown, and purple,—the last is the prevailing colour at this season, the heather being in bloom: nor are the hills monotonous, if he looks at them with the eye of a sportsman, for by this time (we suppose him to have been shooting all the morning) he will have performed many feats, or at any rate will have met with several adventures, and the ground before him is the field of his fame. He now looks with interest on many a rock, and cliff, and hill, which lately appeared but as one of so many ‘crags, knolls, and mounds confusedly hurled!’ He contemplates the site of his achievements, as a general surveys a field of battle during an interval of strife; the experience of the morning has taught him a lesson, and he plans a fresh campaign for the afternoon, or the morrow, or probably the next season, should the same hills be again destined to be the scene of his exploits. The shooter looks down on the other hand from his rocky summit, and, in the bright relief, through the white rents in the clouds, sees the far-off meadows and hamlets, the woods, the rivers, and the lake. He rises, and renews his task. The invigorating influence of the bracing wind on the heights, lends the sportsman additional strength—he puts forth every effort, every nerve is strained—he feels an artificial glow after nature is exhausted, and returns to the cot where he had previously spent a sleepless night, to enjoy his glass of grog, and such a snooze as the citizen never knew!” This is a graphic and true picture of the outset of grouse-shooting; but it is but one amongst many of the exciting situations and
- 66. picturesque positions which this fine sport presents. There is a wide difference, too, between the grouse-shooting of the north of England and of the Highlands. On the English moors, the majority of shooters who assemble there, are the friends or acquaintances of the proprietors, or of their friends and acquaintances, who have received invitations, or procured the favour to shoot for a day or two at the opening of the season. The outbreak on the morning of the 12th, is therefore proportionably multitudinous and bustling. The throng of the people on the preceding evening, crowded into the inns and cottages in the neighbourhood where the best shooting lies, is often amazing. Many sportsmen, who on other occasions would think scorn to enter such a hovel, or jostle in such a crowd, may be seen waiting in patient endurance, in a situation in which a beggar would not envy them. Others will be seen stretched on their cloaks on the floor, while their dogs are occupying their beds, or the soft bottom of a huge old chair; their great anxiety being, to have their dogs fresh and able for the coming day. At the faintest peep of dawn, which is about three o’clock at that season, loud is the sound of guns on all sides, going off farther and farther in the distance. At noon, on some picturesque and breezy hill, you may see a large party congregated to luncheon, where provisions and drink have been conveyed by appointment. There, ten or a dozen sportsmen seated on the ground, all warm in body and mind—their dogs watching eagerly for their share of the feast, which is thrown them with liberal hand— their guns reared against some rock—their game thrown picturesquely on the moorland turf—Flibbertigibbets, with their asses who have brought up the baskets of provisions, the keg of beer, and bottles of porter, are running about and acting the waiters in a style of genuine originality; while keepers and markers are at once lunching and keeping an eye on the dogs, lest they are too troublesome to their masters; who are all talking together with inconceivable ardour of their individual achievements. The situation, the mixture of men and animals, of personages and costumes, all go to make up a striking picture. On the English moorlands, however, grouse-shooting is but as it were a brilliant and passing flash. As the enjoyment of the sport is generally a matter of grace and friendship,
- 67. and is sought by numbers who can only devote to the excursion, at the best, a few days, it is a scene of animation and havoc for a week or ten days, and then its glory is over. During this time, however, the keepers on many estates make a rich harvest, by presents from gentlemen for attendance and guidance to the best haunts of the game—by the loan of dogs at good interest to such as have not come well provided, or have met with accidents, or whose dogs, as is sometimes the case, unused to this kind of sport and scenery, have bolted and disappeared at the first general discharge of guns; and by furnishing, sub rosâ, grouse at a guinea a brace to certain luckless braggadocios, who have boastingly promised to various friends at home plenty of game from the moors; and have not been able to ruffle a single feather! In the Highlands the scene is different. The grounds are more generally rented by individuals or parties; they are wider and wilder, and both from their extent and distance from the populous districts of England are more thinly scattered with shooters. There, some of the sportsmen take their families to their cottages on their shooting-ground, and on which they have probably bestowed some trouble and expense, to render them sufficiently comfortable and convenient for a few months’ occasional summer sojourn, and what in nature can afford a more delicious change from the ordinary course and place of life? Up far amongst the wild mountains and moorlands, amid every fresh and magnificent object—amid fairyland glens of birch and hills of pine, the sight of crystal, rapid, sunny streams, and the sound of waterfalls, in the lands of strange and startling traditions. To intelligent children full of the enjoyment of life and healthful curiosity, in such scenery every thing is wonderful and delightful; to ladies of taste, such a life for a brief season must be equally pleasant. There are some ladies, indeed, of the highest rank, who are in the regular habit of spending a certain portion of every year in the Highlands; and one in particular, of ducal rank, who at that season rambles far and wide amongst the cottages and the beautiful scenery of her native hills, telling her daughters, that if they there indulge in English luxuries, they must prepare them themselves,— such is the simplicity of her mountain residence and establishment;
- 68. and they take their Cook’s Oracle, and wonderfully enjoy the change. The language and costume of the inhabitants are those of a foreign country; every object has its novelty, and the little elegancies of books, music, and furniture, which can be conveyed to such an abode, strike all the more from the stern nature without. Then there is the finest fishing in the lochs and mountain-streams, the most delightful sailing in many places, and in the woods there are the shy roebuck and sometimes the red-deer to be pursued. The grouse and black-cock shooting season is, therefore, longer and steadier there; but the full perfection of its enjoyment is to be found, perhaps, after all, only by the happy mortal who makes one of the select party collected at one of the great Highland houses of the aristocracy, where the best shooting, every requisite of horses, dogs, attendants, etc., are furnished—and where, after the fatigues of the day, the sportsman returns to his own clean room, to an excellent dinner, music, and refined society. But, amid all these seductions, nothing will make the thorough English sportsman forget the first of September. Back he comes, and enters on that regular succession of partridge, pheasant, woodcock, snipe, and wild-fowl shooting, of hunting and coursing, which diversify and fill up the autumn and winter of English rural life. To these pleasures then we leave him. A WORD WITH THE TOO SENSITIVE. I have not attempted to defend the hunter, the courser, or even the shooter, in the preceding chapter, from the charge of cruelty which is perpetually directed against them—they are a sturdy, and now a very intelligent people; often numbering amongst them many of our principal senators, authors, and men of taste, and very capable of vindicating themselves; but I must enact the shield- bearer for a moment, for that very worthy and much-abused old man, Izaak Walton, and the craft which he has made so fashionable. Spite even of Lord Byron’s jingle about the hook and gullet, and a stout fish to pull it, they may say what they will of the old man’s cruelty and inconsistency—the death of a worm, a frog, or a fish, is the height of his infliction, and what is that to the ten thousand
- 69. deaths of cattle, sheep, lambs, fish, and fowl of all kinds, that are daily perpetrated for the sustenance of these same squeamish cavillers! They remind me of a delicate lady, at whose house I was one day, and on passing the kitchen door at ten in the morning, saw a turkey suspended by its heels, and bleeding from its bill, drop by drop. Supposing it was just in its last struggles from a recent death- wound, I passed on, and found the lady lying on her sofa overwhelmed in tears over a most touching story. I was charmed with her sensibility; and the very delightful conversation which I held with her, only heightened my opinion of the goodness of her heart. On accidentally passing by the same kitchen door in the afternoon, six hours afterwards, I beheld, to my astonishment, the same turkey suspended from the same nail, still bleeding, drop by drop, and still giving an occasional flutter with its wings! Hastening to the kitchen, I inquired of the cook, if she knew that the turkey was not dead. “O yes, sir,” she replied, “it won’t be dead, may-happen, these two hours. We always kill turkeys that way, it so improves their colour; they have a vein opened under the tongue, and only bleed a drop at a time!” “And does your mistress know of this your mode of killing turkeys?” “O yes, bless you sir, it’s our regular way; missis often sees ’em as she goes to the gardens—and she says sometimes, ‘Poor things! I don’t like to see ’em, Betty; I wish you would hang them where I should not see ’em!’” I was sick! I was dizzy! It was the hour of dinner, but I walked quietly away, And ne’er repassed that bloody threshold more! I say, what is Izaak Walton’s cruelty to this, and to many another such perpetration on the part of the tender and sentimental? What is it to the grinding and oppression of the poor that is every day going on in society,—to the driving of wheels and the urging of steam- engines, matched against whose iron power thousands daily waste their vital energies? What is it to the laying on of burdens of expense and trouble by the exactions of law, of divinity, of custom,—burdens grievous to be borne, and which they who impose them, will not so much as touch with one of their little fingers?
- 70. They sit at home and turn an easy wheel, And set sharp racks to work to pinch and peel.—John Keats. These things are done and suffered by human beings, and then go the very doers of these things, and cry out mightily against the angler for pricking the gristle of a fish’s mouth! I do not mean to advocate cruelty—far from it! I would have all men as gentle and humane as possible; nor do I argue that because the world is full of cruelty, it is any reason that more cruelty should be tolerated: but I mean to say, that it is a reason why there should not be so much permission to the greater evils, and so much clamour against the less. Is there more suffering caused by angling than by taking fishes by the net? Not a thousandth,—not a ten thousandth part! Where one fish is taken with a hook, it may be safely said that a thousand are taken with the net: for daily are the seas, lakes, and rivers swept with nets; and cod, haddock, halibut, salmon, crabs, lobsters, and every species of fish that supplies our markets, are gathered in thousands and ten thousands—to say nothing of herrings and pilchards by millions. Over these there is no lamentation; and yet their sufferings are as great—for the suffering does not consist so much in the momentary puncture of a hook, as in the dying for lack of their native element. Then go these tender- hearted creatures and feast upon turtles that have come long voyages nailed to the decks of ships in living agonies; upon crabs, lobsters, prawns, and shrimps, that have been scalded to death; and thrust oysters alive into fires; and fry living eels in pans, and curse poor anglers before their gods for cruel monsters, and bless their own souls for pity and goodness, forgetting all the fish-torments they have inflicted! “Ay, but”—they turn round upon you suddenly with what they deem a decisive and unanswerable argument—“Ay, but they cannot approve of making the miseries of sentient creatures a pleasure.” What! is there no pleasure in feasting upon crabs that have been scalded, and eels that have been fried alive? In sucking the juices of an oyster, that has gaped in fiery agony between the bars of your
- 71. kitchen grate? But the whole argument is a sophism and a fallacy. Nobody does seek a pleasure, or make an amusement of the misery of a living creature. The pleasure is in the pursuit of an object, and the art and activity by which a wild creature is captured, and in all those concomitants of pleasant scenery and pleasant seasons that enter into the enjoyment of rural sports;—the suffering is only the casual adjunct, which you would spare to your victim if you could, and which any humane man will make as small as possible. And over what, after all, do these very sensitive persons lament? Over the momentary pang of a creature, which forms but one atom in a living series, every individual of which is both pursuing and pursued, is preying, or is preyed upon. The fish is eagerly pursuing the fly, one fish is pursuing the other, and so it is through the whole chain of living things; and this is the order and system established by the very centre and principle of love, by the beneficent Creator of all life. The too sensitively humane, will again exclaim—“Yes, this is right in the inferior animals: it is their nature, and they only follow the impulse which their Maker has given them.” True; but what is right in them, is equally right in man;—the argument applies with double force in his case. For, is there no such impulse implanted in him? Let every sportsman answer it; let the history of the world answer it; let the heart of every nine-tenths of the human race answer it. Yes, the very fact that we do pursue such sports, and enjoy them, is an irrefragable answer. The principle of chase and taking of prey, which is impressed on almost all living things, from the minutest insect to the lion of the African desert, is impressed with double force on man. By the strong dictates of our nature, by the very words of the Holy Scriptures, every creature is given us for food; our dominion over them, is made absolute. The amiable Cowper asserted that dogs would not pursue game, if they were not taught to do so. We admit the excellent nature of the man, but every day proves that, in this instance, he was talking beyond his knowledge. Every one who knows anything of dogs, knows, that if you bring them up in a town, and keep them away from the habits of their own class to their full growth, the moment they get into the country they will pursue each their peculiar game, with the utmost avidity, and after their own
- 72. Columella De Re Rustica. manner. There is then, unquestionably, an instinctive propensity in one animal to prey upon another—in man pre-eminently so—and it is not the work of wisdom to quench this tendency, but to follow it with all possible gentleness and humanity. CHAPTER V. SCIENTIFIC FARMING. Res rustica, sine dubitatione, proxima, et quasi consanguinea Sapientiæ est. Oh, blessed, who drinks the bliss that Hymen yields, And plucks life’s roses in his quiet fields.—Ebenezer Elliot. There may be a difference of opinion as to the strict utility or wisdom of the pursuits noticed in the last chapter;—of the excellence and rationality of those which form the subject of this, there can be none. Nothing can be more consonant to nature, nothing more delightful, nothing more beneficial to the country, or more worthy of any man, than the Georgical occupations which form so prominent a feature in the rural life of England. Whether a country gentleman seek profit or pleasure in them, he can, at any time, find them. While he is increasing the value of his estate, he is in the midst of health, peace, and a series of operations which have now become purely scientific, and have called in to their accomplishment various other sciences and arts. In every age of the world agricultural pursuits have formed the delight of the greatest nations and the noblest men. Some of the most illustrious kings and prophets of Israel were taken from the fold or the plough. David and Elisha are great names in the history of rural affairs. King Uzziah “built towers in the desert, and digged many wells, for he had much cattle both in the low country and in the plains; husbandmen also, and vinedressers in the mountains, and in Carmel, for he loved
- 73. husbandry.” How delightful are the associations which the literature of Greece and Rome has thrown around country affairs! Homer, Hesiod, and Theocritus—how elysian are the glimpses they give us into rural life! how simple, how peaceful, how picturesque! Laertes, that venerable old monarch, pruning his vines, and fetching young stocks from the woods for his fences. Eumeus, at his rustic lodge, entertaining his prince and his king. Hesiod himself, wandering at the feet of Helicon, less impressed with the sublimity of the poet than with the spirit of the husbandman! He shews us the very infancy of agriculture: Forget not when you sow the grain, to mind That a boy follows with a rake behind; And strictly charge him, as you drive, with care The seeds to cover, and the birds to scare. Works and Days, B. 2. The harrow, an implement well known to King David, for he put the subjected Ammonites under it, was unknown then in Greece! They raked in the grain. That was but the second stage in the progress of tillage; the first undoubtedly being that in which their plough was a pointed stick, and their harrow a bush; as the most ancient drawing of hay-forks shews that they were forked sticks cut from the thicket. But to leave those primitive times of Greece,—there is no nation that at once acquired so vast a military renown and yet retained such a passion for the peaceful pursuits of agriculture as Rome. Nothing is so soon familiarized to the mind of the school-boy as the fact of their generals, dictators, and emperors tilling their own lands—leaving them with reluctance for state honours, and retiring to them with gladness to end their days in meditative tranquillity. Cicero tells us that couriers were first introduced by them, to run between the capitol and their farms, that they themselves might leave them only on most important occasions. Almost every one of their writers on rural affairs, whose works have reached us, were men of distinction in the state. Varro was consul; Cato, the most remarkable man of his time, filled the highest offices; Columella and Palladius were men of note; and Pliny, a patrician officer, was
- 74. governor of Spain. But what is more remarkable even is, that such men as Virgil, Horace, and Cicero, men of imaginative genius, and so involved in court life, or the business of government, should be such passionate lovers of rural concerns. Everyone knows how their writings overflow with the praises of country life, and what delight they took in their farms and villas. Cicero seems as though he could never have done with telling us of the pleasure he took in farming. “I might expatiate,” he says, “on the beauty of verdant groves and meadows, on the charming aspects of vineyards and olive-yards, but to say all in one word, there cannot be a more pleasing, or a more profitable scene than that of a well-cultivated farm. In my opinion, indeed, no kind of occupation is more fraught with happiness, not only as the business of husbandry is of singular utility to mankind, but, as I have said, being attended with its own peculiar pleasures. I will add too, as a further recommendation, and let it restore me to the good graces of the voluptuous, that it supplies both the table and the altar with the greatest variety and abundance. Accordingly, the magazines of the skilful and industrious farmer are plentifully stored with wine and oil, with milk, cheese, and honey; as his yards abound with poultry, and his fields with flocks and herds of kids, lambs, and porkets. The garden also furnishes him with an additional source of delicacies, in allusion to which the farmers pleasantly call a certain piece of ground allotted to that particular use, their dessert. I must not omit, likewise, that in the intervals of their more important business, and in order to heighten the relish of the rest, the sports of the field claim a share of their amusements. * * * Of country occupations I profess myself a warm admirer. They are pleasures perfectly consistent with every degree of advanced years, as they approach the nearest of all others to those of the purely philosophical kind. They are derived from observing the nature and properties of their own earth, which yields a ready obedience to the cultivator’s industry, and returns with interest what he deposits in her charge.”—De Senectute. He then goes on to tell us what delight he took in the cultivation of the vine; in watching the springing and progress of corn; the green blade pushing forth, shooting into a knotted stem, nourished
- 75. and supported by the fibres of the root, terminated in the ear in which the grain is lodged in regular order, and defended from the depredations of birds by its bearded spikes. He tells us that he could name numbers of his most distinguished friends and neighbours, and some of them at very advanced ages, who take such interest in all that is going on at their farms, that they will be present at every important agricultural operation—many of them engaged in improvements of which they will see neither the benefit nor the end. “And what,” says he, “do these noble husbandmen, when they are asked for what purpose they dig and plant, reply,—‘In obedience to the immortal gods, by whose bountiful providence we received these fields from our ancestors, and whose will it is that we should deliver them down with improvement to posterity!’” And this generous and high sense of duty it was which animated the Romans during the better portion of their republic, and kept alive their virtue and their simplicity of life, so far as to give them power to despise wealth, and to command the fortunes of other men. Cicero is delighted with this noble principle, and he reverts with enthusiasm to the picture of Manlius Curius, who, after having conquered the Samnites, the Sabines, and even Pyrrhus himself, passed the honourable remainder of his age in cultivating his farm. He adds, “I can never behold his villa without reflecting with the highest degree of admiration both on the singular moderation of his mind, and the general simplicity of the age in which he flourished. Here it was, while sitting by his fireside, that he nobly rejected the gold which was offered him on the part of the Samnites, and rejected it with this memorable saying, ‘that he placed his glory, not on the abundance of his own wealth, but in commanding those amongst whom it abounded.’” With equal exultation he refers to the enthusiasm into which Xenophon in his treatise of Œconomics breaks forth in the praise of agriculture, and relates the interview of Lysander, the Spartan ambassador, with Cyrus the younger, as told by Socrates to his friend Critobulus, in which Cyrus assures Lysander that all the trees, shrubs, etc., which he admired in his garden, were planted by his own hand. But if such were the charms which agriculture had for the Roman nobility, how much greater ought it to possess for the nobles and
- 76. gentlemen of England! Amid all the advantages and recreations which have been pointed out in the preceding chapters as surrounding the country life of modern England, that of scientific farming is certainly one of the greatest. It is a pursuit full of interest and variety, at once natural, philosophical, and dignified. It is difficult to imagine a man of wealth and education more usefully or honourably employed than in directing the culture and improvement of his estate. Agriculture is now become, indeed, as Cicero termed it in his day, “the nearest of all employments to the purely philosophical kind.” It is a science which requires a first-rate education to prosecute it to its full capability, to make the other arts and sciences of modern times bear upon it, and co-operate with it, so as to add something to its progression, or even to apply beneficially the knowledge of its already established principles and practices.[1] It is no longer an occupation which requires a man to forego the refined pleasures of society, to bury himself amid woods and wildernesses in some obscure hamlet far from the enjoyments and intelligence of the world. As we have already seen, locate himself where he will in these islands, the arts, the elegances, the news and knowledge of civilized life, will penetrate to him by swift agencies, and give him all the real advantages of the city in the peace and fulness of his retirement. And what a noble art is agriculture now become! Look at the manner it is now practised by the most skilful of its professors. Let any one just turn over the leaves of Mr. Loudon’s Encyclopædia of Agriculture, and trace the progress of its implements only, from the plough of the ancients in the shape of a mere pick, to the almost endless machines which the active brains of men and their advancing knowledge of mechanics have given to the scientific farmer. Let any one turn to the list of engravings of farming apparatus in the same excellent work, amounting to about 300, and he will obtain some idea of the amount of science and invention now devoted to the use of the agriculturist. There are no men who have availed themselves of the progress of the arts and of general knowledge more than they. Mechanics, chemistry, hydraulics, steam, all have been seized upon, to develope the principles, or facilitate the operations of agriculture. Within the
- 77. last century the strides which have been made in this interesting department of knowledge are admirable. The Netherlands may be said to have been the mother of our modern agriculture—Scotland its nurse. Tull’s system of horse-hoeing and drill husbandry has been introduced by Dawson, and has brought after it a numerous train of drills, dibbling-machines, horse-hoes, ploughs, rollers, scufflers, scarifiers, watering-machines, brakes, drill-harrows, etc., which we now see almost everywhere where the old system of plain ploughing, harrowing, and broad-cast sowing prevailed to the infinite loss of seed and growth of weeds. Then comes the thrashing machine invented by Menzies, and improved by Meikle from stage to stage, successively adapted to horses, wind, water, and eventually the giant power of steam, thus giving to the operations of the barn a rapidity equal to the skill and neatness displayed in the field. The scientific genius of Sir Humphry Davy, Thompson, Fourcroy, Parmentier, Kirwan, Gay Lussac, and many other eminent chemists, have been employed to investigate more accurately the real nature of soils and manures, and a vast increase of productive power has been the result. Bones, a source of fertility till of late entirely wasted, have done wonders; rape-dust, malt-dust, oil, fish, salt, wood and peat ashes, soot, gypsum, and many other substances, have been made the active agents of human subsistence. The best mixture of crops has been determined by numerous experiments; and the benefits of stall-feeding clearly demonstrated. Mangel- würzel, trifolium incarnatum—a plant which from its rich crimson hue would be an ornament of our fields even were it not a profitable production—and other vegetables, have been added to that plenteous growth of clover, dills, lucerne, rape, turnips, etc., with which modern tillage has enriched both summer and winter stalls. The improvement of the breed of cattle and sheep by Bakewell of Dishly, and the Culleys; the growth of finer and better wools by the introduction and crossing with the Merino by Lord Somerville and others, have been as remarkable as the superior cultivation of the soil. The science of draining has found devotees equally ardent, and has produced the most striking consequences. In many instances the mere act of draining has quadrupled the produce of land. In the
- 78. [1] weald of Kent, land which produced only a rental of five shillings an acre, has been raised by this process to five-and-twenty. And all these objects have been watched over, canvassed, and stimulated by the establishment of agricultural societies, agricultural journals and newspapers, and ploughing matches. Agricultural associations are now to be found in almost every county, and in different districts of the same county, which offer premiums on the best specimens of horses, cattle, and sheep; the best ploughing, and the most steady and industrious farm and household servants. It is a new feature in rural life, to see the whole farming population of a district hastening on a given day, gentlemen, farmers, and farm-servants all in their best array, to some one spot where the cattle are shewn, the ploughing is done, the prizes are awarded by umpires chosen from the most skilful, and the different parties then going to a good dinner, and a long talk and hearty toasting of all the interests of agriculture. This education is now likely to be extended to the great body of farmers. In Ireland, at Templemoyle, a college is established where the sons of farmers are instructed in every branch of science which can enable them to pursue agriculture successfully, while they daily work certain hours on the farm attached, thus making a familiar practical acquaintance with all the best processes of cultivation under the ablest professors. Similar colleges are also contemplated for England. It is really too, as curious to see on our scientific farms the vast variety of implements and machines which these causes have produced;—ploughs—about a dozen and a half swing-ploughs, and upwards of a dozen wheel-ploughs of different constructions, and by different patentees; harrows, drills, cultivators. Every species of soil and crop has its peculiar apparatus; in the field and the farm-yard; for getting seed into the ground, clearing and dressing when there, for thrashing it out and cleaning it for market; for sowing peas, beans, turnips, carrots, parsnips, etc., for chopping, slicing, and preparing them for cattle; their machines for tedding hay, for stacking it with least possible risk, for cutting and steaming it; for ploughing up weeds, ploughing up moorlands, and even roads; for reaping by wholesale, and raking by wholesale; for tapping deep
- 79. springs, and guttering the surface for the escape of top-water; there are their machines for paring and levelling lumpy lands; for cross- cutting furrows to make rough mossy land take seed better; their channels, sluices, and schemes for irrigation. And then, who shall tell all their implements for hay-binding, rope-twisting, furze-pounding for cattle; their novel churns, their ratteries, their new-fangled mole- traps, their poultry-feeders, and pheasant-feeders, by which those birds are enabled to help themselves from tin boxes supplied with grain for them, without feathered depredators being able to go shares with them. Truly Solomon might say that men now-a-days have sought out many inventions! But who shall calculate all the thoughts and the labours of such men as Fitzherbert, Tusser, Gooch, Platt, Hartlib, Weston, Markham, Sir Walter Raleigh, Sir John Norden, John Evelyn, Worlidge, Stillingfleet, Harte, Arthur Young, Maxwell, Lord Kaimes, Sir John Sinclair, etc. etc.? Who shall aggregate and estimate the numerous and valuable suggestions and articles of anonymous writers in the journals; and the personal labours and fostering influence of such men as the late Dukes of Buccleugh, and of Bedford, the Duke of Portland, Earl Spencer, the late Lord Somerville, Mr. Coke of Holkham, now the Earl of Leicester, and many other noblemen and gentlemen who have spent their lives in the unostentatious but most meritorious endeavour to perfect the agricultural science of England? With the exception of naturalists, there are no men whose pursuits seem to me to yield them so much real happiness as intelligent agriculturists whose hearts are in the business; and though there are men whose offices or professions place them more in the public eye, there are none who are more truly the benefactors of their country. Such were Lord Somerville and the Duke of Buccleugh, as described by Sir Walter Scott; and there is a passage in his memoir of the latter nobleman well worth the notice of those who propagate or believe in the nonsense of the economists on the non-influence of absenteeism. “In the year 1817, when the poor stood so much in need of employment, a friend asked the Duke why his Grace did not prepare to go to London in the spring? By way of answer, the Duke shewed him a list of day-labourers then employed in improvements
- 80. on his different estates, the number of whom, exclusive of his regular establishments, amounted to nine hundred and forty-seven persons. If we allow to each labourer two persons, whose support depended on his wages, the Duke was in a manner foregoing, during this severe year, the privilege of his rank, in order to provide with more convenience for a little army of nearly three thousand persons, many of whom must otherwise have found it difficult to obtain subsistence. The result of such conduct is twice blessed; both in the means which it employs, and in the end which it attains in the general improvement of the country. This anecdote forms a good answer to those theorists who pretend that the residence of proprietors on their estates is a matter of indifference to the inhabitants of that district. Had the Duke been residing, and spending his revenue elsewhere, one half of these poor people would have wanted employment and food; and would probably have been little comforted by any metaphysical arguments upon population, which could have been presented to their investigation.”—Scott’s Prose Works, vol. 4. Many such things may be daily heard of the present Duke of Portland, in the neighbourhood of Welbeck Abbey, in Nottinghamshire; which convince you that he is one of those men that contrive to pass through life without much noise, but reaping happiness and respect in abundance, and while gratifying the taste for rural occupation, conferring the most lasting benefits upon the country. I shall close this section of this chapter with the substance of one such act, related to me some years ago. In the manner of relation it may therefore differ somewhat from that in which originally told, but in fact I believe it to be perfectly correct. The Duke found that one of his tenants, a small farmer, was falling, year after year, into arrears of rent. The steward wished to know what should be done. The Duke rode to the farm; saw that it was rapidly deteriorating, and the man, who was really an experienced and industrious farmer, totally unable to manage it, from poverty. In fact, all that was on the farm was not enough to pay the arrears. “John,” said the Duke, as the farmer came to meet him as he rode up to the house, “I want to look over the farm a little.” As they went along,
- 81. —“Really,” said he, “every thing is in very bad case. This won’t do. I see you are quite under it. All your stock and crops won’t pay the rent in arrear. I will tell you what I must do. I must take the farm into my own hands. You shall look after it for me, and I will pay you your wages.” Of course there was no saying nay,—the poor man bowed assent. Presently there came a reinforcement of stock, then loads of manure,—at the proper time, seed, and wood from the plantations for repairing gates and buildings. The Duke rode over frequently. The man exerted himself, and seemed really quite relieved from a load of care by the change. Things speedily assumed a new aspect. The crops and stock flourished; fences and outbuildings were put into good order. In two or three rent days, it was seen by the steward’s books that the farm was paying its way. The Duke on his next visit, said, “Well, John, I think the farm does very well now. We will change again. You shall be tenant again; and as you now have your head fairly above water, I hope you will be able to keep it there.” The Duke rode off at his usual rapid rate. The man stood in astonishment; but a happy fellow he was, when on applying to the steward he found that he was actually re-entered as tenant to the farm just as it stood in its restored condition;—I will venture to say, however, that the Duke himself was the happier man of the two.
- 82. CHAPTER VI. PLANTING. “Jock, when ye hae naething else to do, ye may be aye sticking in a tree; it will be growing, Jock, when ye’re sleeping.”—Heart of Mid-Lothian. What we have just said of the pleasures and benefit of scientific farming, may be said also of planting; it is but another interesting mode of employing time by landed proprietors, at once for recreation and the improvement of their estates. What, indeed, can be more delightful than planning future woods, where, perhaps, now sterile heather, or naked declivities present themselves; clothing, warming, diversifying in imagination your vicinity; then turning your visions into realities, and watching the growth of your forests? Since John Evelyn wrote his eloquent Sylva, and displayed the deplorable condition of our woodlands, and since Dr. Johnson penned his sarcastic Tour to the Hebrides, both England and Scotland have done much to repair the ravages made in the course of ages in our woods. A strong spirit on the subject has grown up in the minds of our landed gentry, and vast numbers of trees of all kinds suitable to our climate have been planted in different parts of the island. The Commissioners of Woods and Forests have made extensive plantations of oak in the New Forest, and other places. In the neighbourhood of all gentlemen’s houses we see evidences of liberal planting: and the rich effect of these young woods is well calculated to strengthen the love of planting. In this part of Surrey, wood, indeed, seems the great growth of the country. Look over the landscape from Richmond Hill, from Claremont, from St. George’s or St. Anne’s Hill, and it is one wide sea of wood. The same is the case in the bordering regions of Buckingham and Berk shires. Richmond Park, Hampton-Court Park, Bushy Park, Claremont and Esher Parks, Oatlands, Painshill, Windsor, Ockham, Bookham—the whole wide country is covered with parks, woods, and fields, the very hedge- rows of which are dense, continuous lines of trees. Look into the
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