Types of Microscopy, Light Microscopes, The Electron Microscope, A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
The earliest microscopes used visible light to create images and were little more than magnifying glasses. Today, more sophisticated compound light microscopes (Figure 4-1) are routinely used in microbiology laboratories. The various types of light microscopy include bright-field, dark-field, fluorescence, and phase contrast microscopy (Figure 4-2). Although each method has specific applications and advantages, bright-field microscopy is most commonly used in introductory classes and clinical laboratories. Many research applications use electron microscopy because of its ability to produce higher quality images of greater magnification.
Light Microscopes
Bright-field microscopy produces an image made from light that is transmitted through a specimen (Figure 4-2A). The specimen restricts light transmission and appears “shadowy” against a bright background (where light enters the microscope unimpeded). Because most biological specimens are transparent, contrast between the specimen and the background can be improved with the application of stains to the specimen (see Sections 5 and 6). The price of improved contrast is that the staining process usually kills cells. This is especially true of bacterial staining protocols. Image formation begins with light coming from an internal or an external light source (Figure 4-3). It passes through the condenser lens, which concentrates the light and makes illumination of the specimen more uniform. Refraction (bending) of light as it passes
4-1 A BINOCULAR COMPOUND MICROSCOPE
A quality microscope is an essential tool for microbiologists.
Most are assembled with exchangeable component parts
and can be customized to suit the particular needs of the user.
4-2 TYPES OF LIGHT MICROSCOPY
A This is a bright-field micrograph of an entire diatom (called a “whole mount”).
Because of its thickness, the entire organism will not be in focus at once.
Continually adjusting the fine focus to clearly observe different levels of
the organism will give a sense of its three around them viewed with dark-field is etween the dimensional structure. The bright rods diatom are bacteria. B The same diatowith dark-field microscopy. Notice thaespecially good at providing contrast borganism’s edge and its interior and the background. Notice also that the bacteria are not visible, though this would not always be the case. C This phase contrast image of the same diatom shows different details of the interior than what is seen in the other two micrographs. Also, notice the bacteria are dark. D This is a fluorescence micrograph of Mycobacterium kansasii. The apple green is one of the characteristic colors of fluorescence microscopy.
4-3 IMAGE PRODUCTION IN A COMPOUND LIGHT MICROSCOPE
Light from the source is focused on the specimen by the condenser lens. It then
enters the objective lens, where it is magnified to produce a real image. The real image is magnified again by the ocular lens to produce a virtual image that is seen by the eye
through the objective lens from the specimen produces a magnified real image. This image is magnified again as it passes through the ocular lens to produce a virtual image that appears below or within the microscope. The amount of magnification produced by each lens is marked on thelens (Figure 4-4A and B). Total magnification of the specimen can be calculated by using the following formula:
4-4 MARKINGS OF MAGNIFICATION AND NUMERICAL APERTURE ON MICROSCOPE COMPONENTS
A Three plan apochromatic objective lenses on the nosepiece of a light microscope. Plan means the lens produces a flat field of view. Apochromatic lenses are made in such a way that chromatic
aberration is reduced to a minimum. From left to right, the lenses magnify 10X, 20X, and 40X, and have numerical apertures of 0.40, 0.70, and 0.85. The 20X lens has other markings on it. The mechanical tube length is the distance from the nosepiece to the ocular and is usually between 160 to
210 mm. However, this 20X lens has been corrected so the light rays are made parallel, effectively creating an infinitely long mechanical tube length (). This allows insertion of accessories into the light path without decreasing image quality. The thickness of cover glass to be used is also given
(0.17 0.01 mm). B A 10X ocular lens. C A condenser (removed from the microscope) with numerical aperture of 1.25. The lever at the right is used to open and close the iris diaphragm and adjust the amount of light entering the specimen.
The practical limit to magnification with a light microscope is around 1300X. Although higher magnifications are possible, image clarity is more difficult to maintain as the magnification increases. Clarity of an image is called resolution (Figure 4-5). The limit of resolution (or resolving power) is an actual measurement of how far apart two points must be in order for the microscope to view them as being separate. Notice that resolution improves as resolving power is made smaller.
The best limit of resolution achieved by a light microscope is about 0.2 µm. (That is, at its absolute best, a light microscope cannot distinguish between two points closer together than 0.2 µm.) For a specific microscope, the actual limit of resolution can be calculated with the following formula:
where D is the minimum distance at which two points can be resolved, is the wavelength of light used, and NAcondenser and NA objective are the numerical apertures of the condenser lens and objective lens, respectively. Because numerical aperture has no units, the units for D are the same as the units for wavelength, which typically are in nanometers (nm).
Numerical aperture is a measure of a lens’s ability to “capture” light coming from the specimen and use it to make the image. As with magnification, it is marked on
4-5 RESOLUTION AND LIMIT OF RESOLUTION The headlights of most
automobiles are around 1.5 m apart. As you look at the cars in the
foreground of the photo, it is easy to see both headlights as separate objects.
The automobiles in the distance appear smaller (but really aren’t) as
does the apparent distance between the headlights. When the apparent
distance between automobile headlights reaches about 0.1 mm, they
blur into one because that is the limit of resolution of the human eye.
the lens (Figures 4-4A and C). Using immersion oil between the specimen and the objective lens increases its numerical aperture and in turn, makes its limit of resolution smaller. (If necessary, oil may also be placed between the condenser lens and the slide.) The result is better resolution.
The light microscope may be modified to improve its ability to produce images with contrast without staining, which often distorts or kills the specimen. In dark-field microscopy (Figure 4-2B), a special condenser is used so only the light reflected off of the specimen enters the objective.
The appearance is of a brightly lit specimen against a dark background, and often with better resolution than that of the bright field microscope.
Phase contrast microscopy (Figure 4-2C) uses special optical components to exploit subtle differences in the refractive indices of water and cytoplasmic components to produce contrast. Light waves that are in phase (that is, their peaks and valleys exactly coincide) reinforce one another and their total intensity (because of the summed amplitudes) increases. Light waves that are out of phase by exactly one-half wavelength cancel each other and result in no intensity—that is, darkness. Wavelengths that are out of phase by any amount will produce some degree of cancellation and result in brightness that is less than maximum but more than darkness. Thus, contrast is provided by differences in light intensity that result from differences in refractive indices in parts of the specimen that put light waves more or less out of phase. As a result, the specimen and its parts appear as various levels of darks and lights.
Fluorescence microscopy (Figure 4-2D) uses a fluorescent dye on the specimen that emits fluorescence when illuminated with ultraviolet radiation. In some cases, specimens possess naturally fluorescing chemicals and no dye is needed.
The Electron Microscope
The electron microscope uses an electron beam to create an image, with electromagnets acting as lenses. The limit of resolution is improved by a factor of 1000 (theoretically down to 0.1 nm, but more realistically down to 2 nm) over the light microscope.
The transmission electron microscope (TEM) (Figure 4-6) produces a two-dimensional image of an ultrathin section by capturing electrons that have passed through the specimen. The degree of interaction between the electrons and the heavy metal stain affects the kinetic energy of the electrons, which are collected by a fluorescent plate. The light of varying intensity emitted from the plate is directly proportional to the electron’s kinetic energy and is used to produce the image. The TEM is useful for studying a cell’s interior, its ultrastructure. A sample transmission electron micrograph is shown in Figure 4-7.
The previous paragraph gave a brief overview of how the TEM works. However, a key to successful transmission electron microscopy is excellent sample preparation. Following is an overview of sample preparation. The specimen is fixed by one of various methods (treatment with formaldehyde, glutaraldehyde, or osmium tetroxide) to prevent cell decomposition, stained with an electron dense material (lead, uranium, or osmium compounds), dehydrated, and embedded in a plastic block (Figure 4-8). It is then cut into thin slices using an ultramicrotome (Figure 4-9) armed with a glass or diamond blade. The slices are captured on a grid
4-6 TRANSMISSION ELECTRON MICROSCOPE
The transmission electron microscope produces an image using electrons that pass
through the specimen. The image is then viewed on the monitor.
This particular model magnifies from 8X up to 630,000X
4-7 TRANSMISSION ELECTRON MICROGRAPH
The TEM produces images of sectioned specimens. Since light is not used, the
image is not in color. These cells were magnified 12,500X
(Figure 4-10), which is inserted into the TEM so it rests in the electron beam path. Figure 4-11 shows what the microscopist sees when working.
A scanning electron microscope (SEM) (Figure 4-12) is used to make a three-dimensional image of the specimen’s surface. In this technique, a beam of electrons is passed over
4-8 TEM SPECIMEN EMBEDDED IN A PLASTIC BLOCK
These plastic resin blocks contain specimens, the black spots within the
blocks. On the right is a trimmed block that has had excess resin cut away to produce a
minute piece of specimen that extends from the block. This is the portion of specimen to
be sectioned (Figure 4-9).
4-9 ULTRAMICROTOME A This ultramicrotome is capable of producing specimen slices 100 nm in thickness (and less). The arm holding the specimen traces an elliptical path as it approaches and is withdrawn from the sample. In each cycle, it is advanced the distance equal to the desired
section thickness, often 100 nm. B The specimen block (S), with the tiny, trimmed down specimen (arrow) facing the blade (Bl), is held in the ultramicrotome chuck. As the specimen moves forward and passes by the glass or diamond blade a thin slice is made, which is caught and floated on
water in the boat (Bt) behind the blade. The specimen holder is withdrawn, returned to the starting position, and advanced by the desired thickness and another cut is made. The process is repeated to produce multiple sections of the same thickness.
4-10 THE GRID AND GRID HOLDER The thin sections are picked up
by a grid (shown), which acts as the equivalent of a glass slide in light
microscopy. The shiny material between grid bars is a plastic film that
fills in the openings and keeps specimens from dropping through. The
grid is placed in a grid holder that is inserted into the TEM. The grid with
its specimens is thus positioned in the electron beam path
4-11 THE VIEWING SCREEN Electron beams do not produce an
image visible to the human eye. In order for the image to be seen, the
microscopist views the specimen on this screen coated with a phosphorescent
material. The kinetic energy of the electrons hitting the
screen is converted to light, which makes the specimen visible. The
thick, dark lines are the grid bars at very low magnification. The image
is also captured by a digital camera and viewed on a computer monitor.
the stained surface of the specimen. Some electrons are reflected (backscatter electrons), whereas other electrons (secondary electrons) are emitted from the metallic stain. These electrons are captured and used to produce the threedimensional image. A sample scanning electron micrograph is shown in Figure 4-13.
As with the TEM, sample preparation involves fixation, dehydration, and staining (but not sectioning). Once the sample is fixed and dehydrated, it is mounted on a stub (Figure 4-14) and coated with the “stain” (usually gold) by a process known as “sputter coating.” A simple explanation of this process is as follows. Argon gas is ionized in an electric field within an evacuated chamber. The positively charged argon ions bombard a gold foil, which releases gold atoms that are free to coat the sample. Figure 4-15 shows a sputter coater.
4-12 SCANNING ELECTRON MICROSCOPE This scanning electron
microscope has the ability to magnify from 12X to 900,000X with a
resolving power as low as 1.0 nm.
4-13 SCANNING ELECTRON MICROGRAPH Like the TEM, the image
produced by the SEM has no color, but it is three-dimensional. This
micrograph is of E. coli.
4-14 SEM SPECIMEN MOUNTED ON A STUB This is a gold-coated
pill bug resting on its back upon the platform of the stub. The larger
cylinder is a holder for the stub
4-15 SPUTTER COATER Stubs with specimens are placed in the
sputter coater chamber, which is then evacuated. Sputtering with
gold occurs when ionized argon gas bombards a gold foil to
release gold atoms. Two specimens are visible within; the purple is
the argon gas.
Suggested Reading
- Michael J. Leboffe & Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
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