Microscope and Microscopy
- 1. MICROSCOPE & MICROSCOPY Principal , Function & Difference of various types of Light & Electron microscope
- 2. MICROSCOPY ⦿Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). ⦿Microscopists explore the relationships between structures & properties for a very wide variety of materials ranging from soft to very hard, from inanimate materials to living organisms, in order to better understand their behaviour.https://www.digistore24.com/redir/35 1558/Mariamkareem/
- 3. INTRODUCTION ⦿in microbiology, the common unit for measuring length is the micrometer(µm), which is equivalent to a millionth (10–6) of a meter. To appreciate how small a micrometer is, consider this: Comparing a micrometer to an inch is like comparing a housefly to New York City’s Empire State Building, 1,472 feet high. Microbial agents range in size from the relatively large, almost visible protozoa (100 µm) down to the incredibly tiny viruses (0.02 µm) ⦿Most bacterial and archaeal cells are about 1 µm to 5 µm in length, although notable exceptions have been discovered recently
- 4. HISTORICAL BACKGROUND Zacharias Janssen 1585 Robert Hooks 1665
- 5. HISTORICAL BACKGROUND Joseph Jackson Lister1830 https://www.digistore24.com/redir/351558/Maria mkaree Antoine van Leeuwenhoek 1677
- 6. INTRODUCTION Achaea & Bacterial cells Empire state building ` 1,472 feet high
- 8. MAGNIFICATION Most microscopes used in microbiology have several objective lenses, including 10* (low power), 40* (high power), and 100* (oil immersion). Most ocular lenses magnify specimens by a factor of 10. Multiplying the magnification of a specific objective lens with that of the ocular, we see that the total magnifications would be 100* for low power, 400* for high power, and 1000* for oil immersion. Some compound light microscopes can achieve a total magnification of 2000* with the oil immersion lens.
- 9. RESOLUTION Resolution (also called resolving power) is the ability of the lenses to distinguish fine detail and structure. Specifically, it refers to the ability of the lenses to distinguish two points that are a specified distance apart. if a microscope has a resolving power of 0.4 nm, it can distinguish two points if they are at least 0.4 nm apart shorter the wavelength of light used in the instrument, the greater the resolution. white light used in a compound light microscope has a relatively long wavelength and cannot resolve structures smaller than about 0.2 μm. Light micrscope resolutionm 1500x van Leeuwenhoek’s tiny spherical lenses had a resolution of 1 μm. human eye are measured at a lateral resolution of 8.55 µm, 576 pixel
- 10. TYPES OF MICROSCOPES ⦿ Microscopy Categories 1: Light (optical) microscopes 2: Electron microscopes Light or optical microscopes further categorized as 1. Polarizing Microscope, 2. Reflected Light Microscope, 3. Bright field microscopy 4. Dark field (Fig. 5a), 5. Phase contrast microscopy 6. Fluorescence microscopy 7. Confocal Microscope 8. SAM 9. Super light resolution microscopy 10. - Multiphoton Microscope – 11. Three-Dimensional Optical Microscopy Electron microcopy is of two types 1. Transmission microscopy and 2. Scanning Electron microscopy
- 11. LIGHT (OPTICAL) MICROSCOPE ⦿ Light travels as wave in crests & troughs. ⦿ The amplitude of the crests & troughs determine the brightness of the light. ⦿ The number of time complete wave occur per unit time is called as frequency and the distance between two consecutive crests is called wavelength (λ) of the light. ⦿ Light microscope wavelength in the range 400-700 nm make up visible spectrum. ⦿ While the UV region consists of wavelengths ranging from 100-385 nm. ⦿ Visualizing any object directly by human eye involves incidence & reflection of light in the visual range. ⦿ Microscopes use day light or light emitted by incandescent bulb. ⦿ Fluorescent & UV microscope employ UV radiations
- 12. LIGHT MICROSCOPE ❖ A modern compound light microscope (LM) has a series of lenses and uses visible light as its source of illumination ❖ We can calculate the total magnification of a specimen by multiplying the objective lens magnification (power) by the ocular lens magnification (power) ❖ Most microscopes used in microbiology have several objective lenses, including 10x (low power), 40x (high power), and 100x (oil immersion) ❖ Most ocular lenses magnify specimens by a factor of 10. Multiplying the magnification of a specific objective lens with that of the ocular, we see that the total magnifications would be 100x for low power, 400x for high power, and 1000x for oil immersion
- 13. CHANGING REFRACTIVE INDEX ⦿ To obtain a clear, finely detailed image under a compound light microscope, specimens must contrast sharply with their medium (the substance in which they are suspended) ⦿ refractive index is a measure of the light-bending ability of a medium. ⦿ To attain such contrast, we must change the refractive index of specimens from that of their medium ⦿ To achieve high magnification (1000*) with good resolution, the objective lens must be small ⦿ To preserve the direction of light rays at the highest magnification, immersion oil is placed between the glass slide and the oil immersion objective lens ⦿ field of vision in a compound light microscope is brightly illuminated. By focusing the light, the condenser produces a brightfield illumination
- 14. HISTORICAL BACKGROUND ⦿Antonie van Leeuwenhoek used first single lens which magnify 300x times in 17th centuary ⦿Robert Hooke, built compound microscopes, which have multiple lenses. ⦿Zaccharias Janssen, is credited with making the first compound microscope around 1600. However, these early compound microscopes were of poor quality and could not be used to see bacteria. ⦿until about 1830 that a significantly better microscope was developed by Joseph Jackson Lister (the father of Joseph Lister).
- 15. HOW TO MAKE IMAGE QUALITY BETTER ⦿ Change contrast of image by changing refractive index in compound microscope. ⦿ Change refractive index of medium and specimen by staining specimen. ⦿ Both have different refractive index so light bend different ⦿ As the light rays travel away from the specimen, they spread out and enter the objective lens, and the image is thereby magnified. ⦿ shorter the wavelength of light used in the instrument, the greater the resolution ⦿ Immersion oil has an index of refraction of 1.5, which is almost identical to the index of refraction of glass
- 20. DARK FIELD MICROSCOPY darkfield microscope is used to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics A darkfield microscope uses a dark field condenser that contains an opaque disc are obscured This technique is frequently used to examine unstained microorganisms suspended in liquid One use for darkfield microscopy is the examination of very thin spirochetes, such as Treponema pallidum (trep-o¯ -NE¯-mah PAL- li-dum), the causative agent of syphilis.
- 22. PHASE-CONTRAST MICROSCOPY ⦿ Phase-contrast microscopy is especially useful because the internal structures of a cell become more sharply defined, permitting detailed examination of living microorganisms ⦿it isn’t necessary to fix (attach the microbes to the microscope slide or stain the specimen— procedures that could distort or kill the microorganisms ⦿One light source and one diffracted source ⦿When brought together make in phase and out- phase ocular image
- 25. DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY ⦿Differential interference contrast (DIC) microscopy is similar to phase-contrast microscopy in that it uses differences in refractive indexes. ⦿DIC microscope uses two beams of light instead of one. ⦿prisms split each light beam, adding contrasting colors to the specimen. Therefore, the resolution of a DIC microscope is higher than that of a standard phase-contrast microscope ⦿he image is brightly colored and appears nearly three-dimensional
- 27. FLUORESCENCE MICROSCOPY ⦿ Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light ⦿ fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis ⦿ . Bacillus anthracis, the causative agent of anthrax, appears apple green when stained with another fluorochrome, fluorescein isothiocyanate (FITC) ⦿ The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent-antibody (FA) technique, or immunofluorescence ⦿ Antibodies and antigen relation ⦿ This technique can detect bacteria or other pathogenic microorganisms, even within cells, tissues, or other clinical specimens
- 29. CONFOCAL MICROSCOPY ⦿ Confocal microscopy is a technique in light microscopy used to reconstruct three-dimensional images. Like fluorescent microscopy, specimens are stained with fluorochromes so they will emit, or return, light. But instead of illuminating the entire field, one plane of a small region of a specimen is illuminated with a short-wavelength (blue) light which passes the returned light through an aperture aligned with the illuminated regi ⦿ two-dimensional images can be obtained, with improved resolution of up to 40% over that of other microscopes. ⦿ The scanned planes of a specimen, which resemble a stack of images, are converted to a digital form that can be used by a computer to construct a three-dimensional representation. ⦿ This procedure makes it possible to image cells up to 1 mm deep in detail
- 32. TWO PHOTONS MICROSCOPY ⦿ Two-photon microscopy uses long-wavelength (red) light, and therefore two photons, instead of one, are needed to excite the fluorochrome to emit light ⦿ longer wavelength allows imaging of living cells in tissues up to 1 mm (1000 μm) deep ⦿ Confocal microscopy can image cells in detail only to a depth of less than 100 mm ⦿ Additionally, the longer wavelength is less likely to generate singlet oxygen, which damages cells ⦿ advantage of TPM is that it can track the activity of cells in real time. For example, cells of the immune system have been observed responding to an antigen.
- 34. SUPER RESOLUTION LIGHT MICROSCOPY ⦿ Maximum resolution for light microscopes was 0.2 μm. However, in 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for the development of a microscope that uses two laser beams ⦿ super-resolution light microscopy, one wavelength stimulates fluorescent molecules to glow, and another wavelength cancels out all fluorescence except for that in one nanometer. ⦿ Cells can be stained with fluorescent dyes that are specific for certain molecules such as DNA or protein, allowing even a single molecule to be tracked in a cell ⦿ . A computer tells the microscope to scan the specimen nanometer by nanometer and then puts the images together
- 36. SCANNING ACOUSTIC MICROSCOPY ⦿Scanning acoustic microscopy (SAM) basically consists of interpreting the action of a sound wave sent through a specimen. A sound wave of a specific frequency travels through the specimen, and a portion of it is reflected back every time it hits an interface within the material. The resolution is about 1 μm. SAM is used to study living cells attached to another surface, such as cancer cells, artery plaque, and bacterial biofilms that foul equipmenhttps://www.digistore24.com/redir/351 558/Mariamkareem/