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2.3: Instruments of Microscopy - Biology

2.3: Instruments of Microscopy - Biology


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Skills to develop

  • Identify and describe the parts of a brightfield microscope
  • Calculate total magnification for a compound microscope
  • Describe the distinguishing features and typical uses for various types of light microscopes, electron microscopes, and scanning probe microscopes

The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses short-wavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope.

Light Microscopy

Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of light microscopes can be used to complement each other in diagnostics and research.

Brightfield Microscopes

The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure (PageIndex{1}); in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective magnification:

[ ext{ocular magnification×objective magnificationocular magnification}; imes; ext{objective magnification}]

For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be

(40×)(10×)=400×

Figure (PageIndex{1}): Components of a typical brightfield microscope.

Components of a typical brightfield microscope.

The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage(a platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses.

When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator, which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than-maximal light levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator.

A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact withchromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses.

At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (Figure (PageIndex{2})). A variety of oils can be used for different types of light.

Figure (PageIndex{2}): (a) Oil immersion lenses like this one are used to improve resolution. (b) Because immersion oil and glass have very similar refractive indices, there is a minimal amount of refraction before the light reaches the lens. Without immersion oil, light scatters as it passes through the air above the slide, degrading the resolution of the image.

Even a very powerful microscope cannot deliver high-resolution images if it is not properly cleaned and maintained. Since lenses are carefully designed and manufactured to refract light with a high degree of precision, even a slightly dirty or scratched lens will refract light in unintended ways, degrading the image of the specimen. In addition, microscopes are rather delicate instruments, and great care must be taken to avoid damaging parts and surfaces. Among other things, proper care of a microscope includes the following:

  • cleaning the lenses with lens paper
  • not allowing lenses to contact the slide (e.g., by rapidly changing the focus)
  • protecting the bulb (if there is one) from breakage
  • not pushing an objective into a slide
  • not using the coarse focusing knob when using the 40⨯ or greater objective lenses
  • only using immersion oil with a specialized oil objective, usually the 100⨯ objective
  • cleaning oil from immersion lenses after using the microscope
  • cleaning any oil accidentally transferred from other lenses
  • covering the microscope or placing it in a cabinet when not in use

Darkfield Microscopy

A darkfield microscope is a brightfield microscope that has a small but significant modification to the condenser. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. The resulting image typically shows bright objects on a dark background (Figure (PageIndex{3}))

Figure (PageIndex{3}): An opaque light stop inserted into a brightfield microscope is used to produce a darkfield image. The light stop blocks light traveling directly from the illuminator to the objective lens, allowing only light reflected or refracted off the specimen to reach the eye.

An opaque light stop inserted into a brightfield microscope is used to produce a darkfield image. The light stop blocks light traveling directly from the illuminator to the objective lens, allowing only light reflected or refracted off the specimen to reach the eye.

Darkfield microscopy can often create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains. For example, thin spirochetes like Treponema pallidum, the causative agent of syphilis, can be best viewed using a darkfield microscope (Figure (PageIndex{4})).

Figure (PageIndex{4}): Use of a darkfield microscope allows us to view living, unstained samples of the spirochete Treponema pallidum. Similar to a photographic negative, the spirochetes appear bright against a dark background. (credit: Centers for Disease Control and Prevention)

Use of a darkfield microscope allows us to view living, unstained samples of the spirochete Treponema pallidum. (credit: Centers for Disease Control and Prevention/C.W. Hubbard)

Exercise (PageIndex{1})

Identify the key differences between brightfield and darkfield microscopy.

Wound infections like Cindy’s can be caused by many different types of bacteria, some of which can spread rapidly with serious complications. Identifying the specific cause is very important to select a medication that can kill or stop the growth of the bacteria.

After calling a local doctor about Cindy’s case, the camp nurse sends the sample from the wound to the closest medical laboratory. Unfortunately, since the camp is in a remote area, the nearest lab is small and poorly equipped. A more modern lab would likely use other methods to culture, grow, and identify the bacteria, but in this case, the technician decides to make a wet mount from the specimen and view it under a brightfield microscope. In a wet mount, a small drop of water is added to the slide, and a cover slip is placed over the specimen to keep it in place before it is positioned under the objective lens.

Under the brightfield microscope, the technician can barely see the bacteria cells because they are nearly transparent against the bright background. To increase contrast, the technician inserts an opaque light stop above the illuminator. The resulting darkfield image clearly shows that the bacteria cells are spherical and grouped in clusters, like grapes.

  • Why is it important to identify the shape and growth patterns of cells in a specimen?
  • What other types of microscopy could be used effectively to view this specimen?

Phase-Contrast Microscopes

Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. To create altered wavelength paths, an annular stop is used in the condenser. The annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens. The objective contains a phase plate containing a phase ring. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate. Because waves have peaks and troughs, they can add together (if in phase together) or cancel each other out (if out of phase). When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness (Figure (PageIndex{5})).

Figure (PageIndex{5}): This diagram of a phase-contrast microscope illustrates phase differences between light passing through the object and background. These differences are produced by passing the rays through different parts of a phase plate. The light rays are superimposed in the image plane, producing contrast due to their interference.

This diagram of a phase-contrast microscope illustrates phase differences between light passing through the object and background. The light rays are superimposed in the image plane, producing contrast due to their interference.

Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy (Figure (PageIndex{6})).

Figure (PageIndex{6}): This figure compares a brightfield image (left) with a phase-contrast image (right) of the same unstained simple squamous epithelial cells. The cells are in the center and bottom right of each photograph (the irregular item above the cells is acellular debris). Notice that the unstained cells in the brightfield image are almost invisible against the background, whereas the cells in the phase-contrast image appear to glow against the background, revealing far more detail.

This figure compares a brightfield image (left) with a phase-contrast image (right) of the same unstained simple squamous epithelial cells. Notice that the unstained cells in the brightfield image are almost invisible against the background, whereas the cells in the phase-contrast image appear to glow against the background, revealing far more detail. (credit: “Clearly kefir”/Wikimedia Commons)

Differential Interference Contrast Microscopes

Differential interference contrast (DIC) microscopes (also known as Nomarski optics) are similar to phase-contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen. In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. Once the beams pass through either the specimen or specimen-free space, they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams. This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens. (Figure (PageIndex{7})).

Figure (PageIndex{7}): A DIC image of Fonsecaea pedrosoi grown on modified Leonian’s agar. This fungus causes chromoblastomycosis, a chronic skin infection common in tropical and subtropical climates.

A DIC image of Fonsecaea pedrosoi grown on modified Leonian’s agar. This fungus causes chromoblastomycosis, a chronic skin infection common in tropical and subtropical climates.

Exercise (PageIndex{2})

What are some advantages of phase-contrast and DIC microscopy?

Fluorescence Microscopes

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) as well as fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI) and acridine orange.

The microscope transmits an excitation light, generally a form of EMR with a short wavelength, such as ultraviolet or blue light, toward the specimen; the chromophores absorb the excitation light and emit visible light with longer wavelengths. The excitation light is then filtered out (in part because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens. This produces an image of the specimen in bright colors against a dark background.

Fluorescence microscopes are especially useful in clinical microbiology. They can be used to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells using fluorescence microscopy based upon whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features.

One of the most important applications of fluorescence microscopy is a technique called immunofluorescence, which is used to identify certain disease-causing microbes by observing whether antibodies bind to them. (Antibodies are protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them.) There are two approaches to this technique: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that the target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach directly to the pathogen.

In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the pathogen, but they do bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier visualize features in the specimen (Figure (PageIndex{8})).

Figure (PageIndex{8}): (a) A direct immunofluorescent stain is used to visualize Neisseria gonorrhoeae, the bacterium that causes gonorrhea. (b) An indirect immunofluorescent stain is used to visualize larvae of Schistosoma mansoni, a parasitic worm that causes schistosomiasis, an intestinal disease common in the tropics. (c) In direct immunofluorescence, the stain is absorbed by a primary antibody, which binds to the antigen. In indirect immunofluorescence, the stain is absorbed by a secondary antibody, which binds to a primary antibody, which, in turn, binds to the antigen. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Centers for Disease Control and Prevention)

(a) A direct immunofluorescent stain is used to visualize Neisseria gonorrhoeae, the bacterium that causes gonorrhea. (b) An indirect immunofluorescent stain is used to visualize larvae of Schistosoma mansoni, a parasitic worm that causes schistosomiasis, an intestinal disease common in the tropics. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Centers for Disease Control and Prevention/Dr. Sulzer)

Exercise (PageIndex{3})

Why must fluorochromes be used to examine a specimen under a fluorescence microscope?

Confocal Microscopes

Whereas other forms of light microscopy create an image that is maximally focused at a single distance from the observer (the depth, or z-plane), a confocal microscope uses a laser to scan multiple z-planes successively. This produces numerous two-dimensional, high-resolution images at various depths, which can be constructed into a three-dimensional image by a computer. As with fluorescence microscopes, fluorescent stains are generally used to increase contrast and resolution. Image clarity is further enhanced by a narrow aperture that eliminates any light that is not from the z-plane. Confocal microscopes are thus very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed (Figure (PageIndex{9})).

Figure (PageIndex{9}): Confocal microscopy can be used to visualize structures such as this roof-dwelling cyanobacterium biofilm. (credit: modification of work by American Society for Microbiology).

Confocal microscopy can be used to visualize structures such as this roof-dwelling cyanobacterium biofilm. (credit: American Society for Microbiology)

Two-Photon Microscopes

While the original fluorescent and confocal microscopes allowed better visualization of unique features in specimens, there were still problems that prevented optimum visualization. The effective sensitivity of fluorescence microscopy when viewing thick specimens was generally limited by out-of-focus flare, which resulted in poor resolution. This limitation was greatly reduced in the confocal microscope through the use of a confocal pinhole to reject out-of-focus background fluorescence with thin (<1 μm), unblurred optical sections. However, even the confocal microscopes lacked the resolution needed for viewing thick tissue samples. These problems were resolved with the development of the two-photon microscope, which uses a scanning technique, fluorochromes, and long-wavelength light (such as infrared) to visualize specimens. The low energy associated with the long-wavelength light means that two photons must strike a location at the same time to excite the fluorochrome. The low energy of the excitation light is less damaging to cells, and the long wavelength of the excitation light more easily penetrates deep into thick specimens. This makes the two-photon microscope useful for examining living cells within intact tissues—brain slices, embryos, whole organs, and even entire animals.

Currently, use of two-photon microscopes is limited to advanced clinical and research laboratories because of the high costs of the instruments. A single two-photon microscope typically costs between $300,000 and $500,000, and the lasers used to excite the dyes used on specimens are also very expensive. However, as technology improves, two-photon microscopes may become more readily available in clinical settings.

Exercise (PageIndex{4})

What types of specimens are best examined using confocal or two-photon microscopy?

Electron Microscopy

The maximum theoretical resolution of images created by light microscopes is ultimately limited by the wavelengths of visible light. Most light microscopes can only magnify 1000⨯, and a few can magnify up to 1500⨯, but this does not begin to approach the magnifying power of an electron microscope (EM), which uses short-wavelength electron beams rather than light to increase magnification and resolution.

Electrons, like electromagnetic radiation, can behave as waves, but with wavelengths of 0.005 nm, they can produce much better resolution than visible light. An EM can produce a sharp image that is magnified up to 100,000⨯. Thus, EMs can resolve subcellular structures as well as some molecular structures (e.g., single strands of DNA); however, electron microscopy cannot be used on living material because of the methods needed to prepare the specimens.

There are two basic types of EM: the transmission electron microscope (TEM) and the scanning electron microscope (SEM)(Figure (PageIndex{10})). The TEM is somewhat analogous to the brightfield light microscope in terms of the way it functions. However, it uses an electron beam from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the specimen onto a detector. Electrons pass through the specimen, and then the detector captures the image (Figure (PageIndex{11})).

Figure (PageIndex{10}): (a) A transmission electron microscope (TEM). (b) A scanning electron microscope (SEM). (credit a: modification of work by “Deshi”/Wikimedia Commons; credit b: modification of work by “ZEISS Microscopy”/Flickr)

Figure (PageIndex{11}): Electron microscopes use magnets to focus electron beams similarly to the way that light microscopes use lenses to focus light.

For electrons to pass through the specimen in a TEM, the specimen must be extremely thin (20–100 nm thick). The image is produced because of varying opacity in various parts of the specimen. This opacity can be enhanced by staining the specimen with materials such as heavy metals, which are electron dense. TEM requires that the beam and specimen be in a vacuum and that the specimen be very thin and dehydrated. The specific steps needed to prepare a specimen for observation under an EM are discussed in detail in the next section.

SEMs form images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons. This can create highly detailed images with a three-dimensional appearance that are displayed on a monitor (Figure (PageIndex{12})). Typically, specimens are dried and prepared with fixatives that reduce artifacts, such as shriveling, that can be produced by drying, before being sputter-coated with a thin layer of metal such as gold. Whereas transmission electron microscopy requires very thin sections and allows one to see internal structures such as organelles and the interior of membranes, scanning electron microscopy can be used to view the surfaces of larger objects (such as a pollen grain) as well as the surfaces of very small samples (Figure (PageIndex{13})). Some EMs can magnify an image up to 2,000,000⨯.1

Figure (PageIndex{12}): These schematic illustrations compare the components of transmission electron microscopes and scanning electron microscopes.

Figure (PageIndex{13}): (a) This TEM image of cells in a biofilm shows well-defined internal structures of the cells because of varying levels of opacity in the specimen. (b) This color-enhanced SEM image of the bacterium Staphylococcus aureus illustrates the ability of scanning electron microscopy to render three-dimensional images of the surface structure of cells. (credit a: modification of work by American Society for Microbiology; credit b: modification of work by Centers for Disease Control and Prevention)

Exercise (PageIndex{5})

  1. What are some advantages and disadvantages of electron microscopy, as opposed to light microscopy, for examining microbiological specimens?
  2. What kinds of specimens are best examined using TEM? SEM?

Using Microscopy to study Biofilms

A biofilm is a complex community of one or more microorganism species, typically forming as a slimy coating attached to a surface because of the production of an extrapolymeric substance (EPS) that attaches to a surface or at the interface between surfaces (e.g., between air and water). In nature, biofilms are abundant and frequently occupy complex niches within ecosystems (Figure (PageIndex{14})). In medicine,biofilms can coat medical devices and exist within the body. Because they possess unique characteristics, such as increased resistance against the immune system and to antimicrobial drugs, biofilms are of particular interest to microbiologists and clinicians alike.

Because biofilms are thick, they cannot be observed very well using light microscopy; slicing a biofilm to create a thinner specimen might kill or disturb the microbial community. Confocal microscopy provides clearer images of biofilms because it can focus on one z-plane at a time and produce a three-dimensional image of a thick specimen. Fluorescent dyes can be helpful in identifying cells within the matrix. Additionally, techniques such as immunofluorescence and fluorescence in situ hybridization (FISH), in which fluorescent probes are used to bind to DNA, can be used.

Electron microscopy can be used to observe biofilms, but only after dehydrating the specimen, which produces undesirable artifacts and distorts the specimen. In addition to these approaches, it is possible to follow water currents through the shapes (such as cones and mushrooms) of biofilms, using video of the movement of fluorescently coated beads (Figure (PageIndex{15})).

Figure (PageIndex{14}): A biofilm forms when planktonic (free-floating) bacteria of one or more species adhere to a surface, produce slime, and form a colony. (credit: Public Library of Science).

Figure (PageIndex{15}): In this image, multiple species of bacteria grow in a biofilm on stainless steel (stained with DAPI for epifluorescence miscroscopy). (credit: Ricardo Murga, Rodney Donlan).

Scanning Probe Microscopy

A scanning probe microscope does not use light or electrons, but rather very sharp probes that are passed over the surface of the specimen and interact with it directly. This produces information that can be assembled into images with magnifications up to 100,000,000⨯. Such large magnifications can be used to observe individual atoms on surfaces. To date, these techniques have been used primarily for research rather than for diagnostics.

There are two types of scanning probe microscope: the scanning tunneling microscope (STM) and the atomic force microscope (AFM). An STM uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen. This current occurs via quantum tunneling of electrons between the probe and the specimen, and the intensity of the current is dependent upon the distance between the probe and the specimen. The probe is moved horizontally above the surface and the intensity of the current is measured. Scanning tunneling microscopy can effectively map the structure of surfaces at a resolution at which individual atoms can be detected.

Similar to an STM, AFMs have a thin probe that is passed just above the specimen. However, rather than measuring variations in the current at a constant height above the specimen, an AFM establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen. As the probe tip is passed over the specimen, forces between the atoms (van der Waals forces, capillary forces, chemical bonding, electrostatic forces, and others) cause it to move up and down. Deflection of the probe tip is determined and measured using Hooke’s law of elasticity, and this information is used to construct images of the surface of the specimen with resolution at the atomic level (Figure (PageIndex{16})).

Figure (PageIndex{16}): STMs and AFMs allow us to view images at the atomic level. (a) This STM image of a pure gold surface shows individual atoms of gold arranged in columns. (b) This AFM image shows long, strand-like molecules of nanocellulose, a laboratory-created substance derived from plant fibers. (credit a: modification of work by “Erwinrossen”/Wikimedia Commons).

Exercise (PageIndex{6})

  1. Which has higher magnification, a light microscope or a scanning probe microscope?
  2. Name one advantage and one limitation of scanning probe microscopy.

Figure (PageIndex{17}): (credit “Brightfield”: modification of work by American Society for Microbiology; credit “Darkfield”: modification of work by American Society for Microbiology; credit “Phase contrast”: modification of work by American Society for Microbiology; credit “DIC”: modification of work by American Society for Microbiology; credit “Fluorescence”: modification of work by American Society for Microbiology; credit “Confocal”: modification of work by American Society for Microbiology; credit “Two-photon”: modification of work by Alberto Diaspro, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola Ramoino, Cesare Usai).

Figure (PageIndex{18}): (credit “TEM”: modification of work by American Society for Microbiology; credit “SEM”: modification of work by American Society for Microbiology)

Figure (PageIndex{19}): Microscopy techniques for scanning probe microscopes.

Key Concepts and Summary

  • Numerous types of microscopes use various technologies to generate micrographs. Most are useful for a particular type of specimen or application.
  • Light microscopy uses lenses to focus light on a specimen to produce an image. Commonly used light microscopes include brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.
  • Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light microscopy. The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.
  • Scanning probe microscopy produces images of even greater magnification by measuring feedback from sharp probes that interact with the specimen. Probe microscopes include the scanning tunneling microscope (STM) and the atomic force microscope (AFM).

Glossary

atomic force microscope
a scanning probe microscope that uses a thin probe that is passed just above the specimen to measure forces between the atoms and the probe
binocular
having two eyepieces
brightfield microscope
a compound light microscope with two lenses; it produces a dark image on a bright background
coarse focusing knob
a knob on a microscope that produces relatively large movements to adjust focus
chromophores
pigments that absorb and reflect particular wavelengths of light (giving them a color)
condenser lens
a lens on a microscope that focuses light from the light source onto the specimen
confocal microscope
a scanning laser microscope that uses fluorescent dyes and excitation lasers to create three-dimensional images
darkfield microscope
a compound light microscope that produces a bright image on a dark background; typically a modified brightfield microscope
diaphragm
a component of a microscope; typically consists of a disk under the stage with holes of various sizes; can be adjusted to allow more or less light from the light source to reach the specimen
differential interference-contrast microscope
a microscope that uses polarized light to increase contrast
electron microscope
a type of microscope that uses short-wavelength electron beams rather than light to increase magnification and resolution
fine focusing knob
a knob on a microscope that produces relatively small movements to adjust focus
fluorescence microscope
a microscope that uses natural fluorochromes or fluorescent stains to increase contrast
fluorochromes
chromophores that fluoresce (absorb and then emit light)
illuminator
the light source on a microscope
immunofluorescence
a technique that uses a fluorescence microscope and antibody-specific fluorochromes to determine the presence of specific pathogens in a specimen
monocular
having a single eyepiece
objective lenses
on a light microscope, the lenses closest to the specimen, typically located at the ends of turrets
ocular lens
on a microscope, the lens closest to the eye (also called an eyepiece)
oil immersion lens
a special objective lens on a microscope designed to be used with immersion oil to improve resolution
phase-contrast microscope
a light microscope that uses an annular stop and annular plate to increase contrast
rheostat
a dimmer switch that controls the intensity of the illuminator on a light microscope
scanning electron microscope (SEM)
a type of electron microscope that bounces electrons off of the specimen, forming an image of the surface
scanning probe microscope
a microscope that uses a probe that travels across the surface of a specimen at a constant distance while the current, which is sensitive to the size of the gap, is measured
scanning tunneling microscope
a microscope that uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen
stage
the platform of a microscope on which slides are placed
total magnification
in a light microscope is a value calculated by multiplying the magnification of the ocular by the magnification of the objective lenses
transmission electron microscope (TEM)
a type of electron microscope that uses an electron beam, focused with magnets, that passes through a thin specimen
two-photon microscope
a microscope that uses long-wavelength or infrared light to fluoresce fluorochromes in the specimen
x-y mechanical stage knobs
knobs on a microscope that are used to adjust the position of the specimen on the stage surface, generally to center it directly above the light

Contributor

  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at https://openstax.org/books/microbiology/pages/1-introduction)


Fluorescence Microscopy, Applications

Principles of Fluorescence Microscopy

Fluorescence microscopy is a technique whereby fluorescent substances are examined in a microscope. It has a number of advantages over other forms of microscopy, offering high sensitivity and specificity.

In fluorescence microscopy, the specimen is illuminated (excited) with light of a relatively short wavelength, usually blue or ultraviolet (UV). The specimen is examined through a barrier filter that absorbs the short-wavelength light used for illumination and transmits the fluorescence, which is therefore seen as bright against a dark background ( Figure 1 ). Because fluorescence is observed as luminosity on a dark background, fluorescent constituents of the specimen can be seen even in extremely small amounts. There are several different modes of fluorescence microscopy, of which the most important is confocal fluorescence microscopy.

Figure 1 . Fluorescence photomicrographs of a section of a plant stem, cut longitudinally (main picture) and transversely (inset, at higher magnification). The tissue was stained with a fluorochrome, Aniline blue, to show the sugar-conducting tissue (phloem). Aniline blue stains specialized regions of the cell walls. Intense fluorescence is most obvious on the transverse (end) walls of elongated cells. The end walls have pores so that there is continuity from cell to cell, forming a continuous tube in which sugars may be moved down the plant from the leaves. A face view of one of the end walls is shown in the inset. There is a ring of fluorescence around each pore. There is also fluorescent staining, to a much lesser degree, on the longitudinal walls. This surrounds pores that allow sugar transport between adjacent tubes. Aniline blue stains 1 → 3β–glucans. Here it is staining callose, a glucan which is deposited to occlude the pores should the tubes become damaged, as when tissue is cut. This presumably blocks the cut part of the tubes, minimizing loss of sugars from the cut ends of the tubes, and may well also minimize entry of microorganisms that would be attracted by leaking sugars. The tubes shown in the main picture are 70 μm in diameter. Field size of main picture approximately 500×1000 μm (0.5×1 mm), of inset approximately 100×100 μm.

Pictures courtesy of Professor AE Ashford, University of New South Wales.

In most modern fluorescence microscopes, epi-illumination is employed. This means that the light used for excitation is reflected onto the specimen through the objective, which acts as a condenser. Opaque or very thick objects can be examined using epi-illumination, even the skin of living people.

The position of fluorescence microscopy in relation to other techniques is summarized in Table 1 . Conventional, transmitted-light, absorption microscopy is appropriate for coloured objects of resolvable size, and instrumentally is the simplest form of microscopy. Colourless, transparent objects can be studied only by retardation techniques (polarization, phase-contrast, interference) these techniques depend upon conversion of phase retardation into changes in intensity that can be seen by the eye. An exception is darkground illumination, which may reveal colourless transparent objects by reflection or refraction at interfaces of differing refractive indices. Darkground microscopy is otherwise suitable mainly for particulate matter, and (like fluorescence microscopy) can reveal the positions of particles too small to be resolved. Fluorescence microscopy is closely allied to transmission (absorption) microscopy in its range of application, but possesses particular advantages: great sensitivity for detection and quantification of small amounts of fluorescent substances or small particles, and the possibility of application to opaque objects. Since fluorescence involves two wavelength bands (excitation and emission), optical specificity can be substantially increased by careful selection of filter combinations to favour the excitation and emission of some particular fluorophore, and modern developments also permit time-resolution of the fluorescence lifetime.

Table 1 . Applicability of fluorescence microscopy, compared with other techniques

Type of microscopy
SpecimenFluorescenceAbsorption (transmission)Retardation (DIC, Pol, etc.)Reflection (incl. darkground)
ColouredSuitableSuitableUnsuitableSuitable
TransparentImpossibleImpossibleSuitableImpossible
OpaqueSuitableImpossibleSuitableSuitable
DynamicSuitableImpossibleImpossibleImpossible
Particles below limit of resolutionSuitableImpossibleUnsuitableSuitable

Absorption microscopy is the conventional transmitted-light type. Retardation microscopy includes Nomarski interference-contrast (DIC), phase-contrast and polarization. Reflection microscopy includes darkground.

Fluorescence microscopy, because of its complexity, gives more difficulty than usual in interpretation of the image. Factors which may affect the appearance of the image in a fluorescence microscope are related to the specimen, to the microscope optical system (particularly the filter combination) and to the observer’s own optical and neurological characteristics. In particular, the use of a narrow-band barrier filter can be misleading, since it makes everything appear its specific colour, whereas a wide-band or long (wavelength)-pass filter allows differentiation of different colours. Even photography, apparently objective, may be misleading if not interpreted correctly.

The current definitive texts on fluorescence microscopy are those of Rost (see Further Reading). There also exist several introductory works, such as that of Abramowicz, and a vast specialized literature. The major texts on confocal fluorescence microscopy are those of Pawley and of Wilson.


USB computer microscopes, also called computer or computer-connected microscopes, plug into a USB port on a computer or television. Instead of looking using an eyepiece, the viewer examines the specimen via the computer monitor or TV screen, like a webcam with a lens. Most of these microscopes are handheld and can save images as files or videos. However, most have only low-level magnification, and adequate illumination can be a problem.

Pocket microscopes are handheld, durable, and useful for field work. Sizes vary, and some are the size of an ink pen. Most use natural light or are battery-powered, with 25x to 100x magnification. Portable microscopes may also be digital.


History of Microscopes

1590: Two Dutch spectacle-makers and father-and-son team, Hans and Zacharias Janssen, create the first microscope.

1667: Robert Hooke's famous "Micrographia" is published, which outlines Hooke's various studies using the microscope.

1675: Enter Anton van Leeuwenhoek, who used a microscope with one lens to observe insects and other specimen. Leeuwenhoek was the first to observe bacteria. 18th century: As technology improved, microscopy became more popular among scientists. Part of this was due to the discovery that combining two types of glass reduced the chromatic effect.

1830: Joseph Jackson Lister discovers that using weak lenses together at various distances provided clear magnification.

1878: A mathematical theory linking resolution to light wavelength is invented by Ernst Abbe.

1903: Richard Zsigmondy invents the ultramicroscope, which allows for observation of specimens below the wavelength of light.

1932: Transparent biological materials are studied for the first time using Frits Xernike's invention of the phase-contrast microscope.

1938: Just six years after the invention of the phase contrast microscope comes the electron microscope, developed by Ernst Ruska, who realized that using electrons in microscopy enhanced resolution.

1981: 3-D specimen images possible with the invention of the scanning tunneling microscope by Gerd Binnig and Heinrich Rohrer.

Origin: The origin of the word microscope according to the Online Etymology Dictionary is as follows: 1656, from Mod.L. microscopium, lit. "an instrument for viewing what is small," from Gk. micro- (q.v.) + -skopion. "means of viewing," from skopein "look at." Microscopic "of minute size" is attested from 1760s.

History of the Compound Microscope

Just as the Greeks had a fully functioning radiant heating system operating two thousand years before those only now being introduced in the US, so the origins of the compound light microscope appear to be traced, not to Holland, England or France - but to China which is perhaps appropriate given the present predominance of China in supplying compound light microscopes!

The Water Microscope

According to an ancient Chinese text, the Chinese viewed magnified specimens through a lens at the end of a tube, which tube was filled with varying levels of water according to the degree of magnification they wished to achieve. Ingenious, effective and repeatable in the home, today. That this occurred some 4,000 years ago in the Chow-Foo dynasty and more than 3,500 years before the "father of modern microscopy" was born is quite remarkable.

That these Chinese ancients achieved magnification levels of 150 times today's standard, or 100 moou, is breath-taking. It is as if they developed a town car that achieved Mach II. If they did build such a car, no reference to it has ever been found. Similarly, there is no further known reference to such a compound microscope device until we come back to the Greeks again.

No less a person than Aristotle describes the workings of a microscope in some detail. The Greeks certainly made good use of curved lenses, which are an essential component of any stereo or compound microscope. Ancient Greek boys probably shared every American boy's sense of triumph of using a curved lens, or magnifying glass, to start a fire. The Greeks, however, also used it for surgical procedures, not on ants as little boys are wont to do, but on people - to cauterize wounds and lesions caused by leprosy and so forth.

Ancient Egyptians and Romans also used various curved lenses although no reference to a compound microscope has been found. The Greeks did, however, give us the word "microscope." It comes from two Greek words, "uikpos," small and "okottew," view. However, while Ancient Chinese, Greeks and Romans all applied their infinite wisdom to the issue, there is no known reference to either the use of artificial light or to multiple lenses. In other words, we can give great credit to the Ancients for their foresight and achievements, but we have to look elsewhere to uncover both the first light and compound microscope.

Incredibly, the next historical references with anything at all to do with microscopes, or more accurately, optics is 1,200 years after Rome was sacked and, even then, the references are only to the use of lenses in the invention of spectacles. Put another way round, some of the smartest people the planet has ever produced, played and worked with single lenses for several thousand years without taking it further.

Spectacles

Then, within just a few short years in Tuscany, Italy, two men claimed to have independently invented spectacles. The evidence? Their tombstones! One, Salvano d'Aramento degli Amati died in 1284 in Florence and claimed to have kept the process secret. The other, Allessandro della Spina died in 1317 and claimed to have revealed his process. Pisa and Florence are but a short gallop away. Coincidence? You decide.

In any event, a local monk, Girodina da Rivalta gave a sermon in 1306 in which he enthusiastically endorsed spectacles as a terrific invention and in passing, indicated that they had been in use for about 20 years. Finally, in 1289, another local from the Popozo family bemoaned that "I am so debilitated by age that without the glasses known as spectacles, I would no longer be able to read or write."

Telescopes

At about the same time, it appears that lenses were being used in early telescopes. In the 13th century, the Englishman, Roger Bacon discusses them at length. Both spectacles and microscopes are relevant to microscopes because they trace the increasingly sophisticated use of lenses - the essential optical component of any microscope.

Then, a mere 200-300 years later, we find a plethora of references and hard evidence of both telescopes and microscopes. The Renaissance had arrived and with it, an abundant flowering in the arts and sciences. Most importantly, with the invention of the printing oress, ideas and developments could be spread easily and rapidly. As a result, Thomas Digges' work on the telescope in England in the mid-16th century and Hans Lippershey's work which included applying for a telescope patent were transmitted to others, including no less a genius than Galileo.

Galileo immediately began to work with lenses. In a short timeframe, he developed an improved telescope with a focusing device and went on to conquer the stars. That said, we should also pay tribute to Sir Isaac Newton who around the same time in the UK, invented the reflecting telescope.

Compound Microscopes

But what of microscopes?Well, the same Hans Lippershey and his son, Zaccharias Hanssen was experimenting with a variety of lenses. In the late 1590?s, they used several lenses in a tube and were amazed to see that the object at the end of the tube was magnified significantly beyond the capability of a magnifying glass. They had just invented the compound microscope. That is to say, they had discovered that an image magnified by a single lens can be further magnified by a second or more lenses.

Then, in the mid 17th century, an Englishman, Robert Hooke and a Dutchman, Anthony Van Leeuwenhoek took the microscope to new levels. Hooke was a sickly genius who loved to experiment. He did so across a huge range of scientific fields of study and with prolific success. He invented the universal joint, the iris diaphragm (another key component of many modern light microscopes), a respirator, an anchor escapement and balance spring for clocks.

He also worked out the correct theory of combustion devised an equation describing elasticity that is still used today ("Hooke's Law") and invented or improved meteorological instruments such as the barometer, anemometer, and hygrometer and so on. Most of all, however, he is known for Micrographia, his studies with a microscope, published in 1665. Micrographia became an overnight sensation not just for what he described but for the superb drawings that he made.

He described a new world alongside exquisite drawings of the stinging hairs on a nettle, a flea and, most famously of all, the honeycomb structure or "cells" of a cork. It was Hooke who coined the term "cells" when describing living tissue. Interestingly, while Hooke did use a compound microscope, he found that it much strained and weakened his sight. For his Micrographia, he preferred to use a simple, single lens microscope made of gold and leather and illuminated by a candle. Perhaps the first light microscope?

Antonie van Leeuwenhoek - the Father of the Microscope

It was Leeuwenhoek, however, who lived at the same time as Hooke and drew on Hooke's work to take microscope design to new levels of sophistication. As a draper, he used a simple microscope to examine cloth. As a scientist, he began to experiment with new ways of grinding lenses in order to improve the optical quality. In total, he ground some 550 lenses, some of which had a linear magnifying power of 500 and a resolving power of one-millionth of an inch - an astounding achievement.

Leeuwenhoek detailed these achievements in almost 200 letters to The Royal Society in London where no less a person than Robert Hooke validated them. The result of all this work was a simple, single lens, hand-held microscope. The specimen was mounted on the top of the pointer, above which lay a convex lens attached to a metal holder. The specimen was then viewed through a hole on the other side of the microscope and was focused using a screw.

Perhaps his most famous experiment came in 1674 when he viewed some lake water:

"I now saw very plainly that these were little eels, or worms, lying all huddled up together and wriggling just as if you saw, with thenaked eye, a whole tubful of little eels and water, with the eels squirming among one another and the whole water seemed to be alive with these multifarious animalcules.

This was for me, among all the marvels that I have discovered in nature, the most marvelous of all and I must say, for my part, that no more pleasant sight has every yet come before my eyes that these many thousand of living creatures seen all alive in a little drop of water, moving among one another, each several creature having its own proper motion."

He had discovered bacteria. He had earned his title of the Father of the Microscope. Interestingly, it took until 1839, nearly two hundred years later, before cells were finally acknowledged as the basic units of life.

18th/19th Centuries

The next major step in the history of the microscope occurred another 100 years later with the invention of the achromatic lens by Charles Hall, in the 1730s. He discovered that by using a second lens of different shape and refracting properties, he could realign colors with minimal impact on the magnification of the first lens.

Then in 1830, Joseph Lister solved the problem of spherical aberration (light bends at different angles depending on where it hits the lens) by placing lenses at precise distances from each other. Combined, these two discoveries contributed towards a marked improvement in the quality of image. Previously, due to the poor quality of glass and imperfect lens, microscopists had been viewing nothing but distorted images - somewhat like the first radios were extremely crackly.

It is worth remembering that up until now, each new stride has been in the quality or application of the lenses. Then, in 1863, one of the several new manufacturers of microscopes, the Ernst Leitz company, addressed a mechanical issue with the introduction of the first revolving turret with no less than five objectives.

This improvement was quickly followed in 1866 when Carl Zeiss recruited Ernst Abbe as his director of research at the Zeiss Optical Works. Abbe laid out the framework of what would become the modern computational optics development approach. He made clear the difference between magnification and resolution and criticized the practice of using eyepieces with too high a magnification as "empty magnification." By 1869, his work produced a new patented illumination device - the Abbe condenser.

Abbe Condenser: Abbe's work on a wave theory of microscopic imaging (the Abbe Sine Condition) made possible the development of a new range of seventeen microscope objectives - three of these were the first immersion objectives and all were designed based on mathematical modeling. As Abbe noted, his creations were "based on a precise study of the materials used, the designs concerned are specified by computation to the last detail - every curvature, every thickness, every aperture of a lens - so that any trial and error approach is excluded."

From here on, microscopes were designed based on sound laws of physics rather than the trial and error that had characterized the pioneers. At the same time, a number of companies set up specialized manufacturing plants focused on manufacturing precision microscopes. Research and development continued to bear fruit.

In 1880, the first microtomes began to be used that enabled significantly thinner samples to be prepared in order to improve sample. In 1893, another Zeiss employee, August Kohler figured out an unparalleled illumination system that is still known as Kohler illumination. Using double diaphragms, the system provides triple benefits of a uniformly illuminated specimen, a bright image and minimal glare. In other words, Kohler achieved an almost perfect image.

The mass market for microscopes had arrived at the same time as precision engineering and it is little wonder that a plethora of stunning results were obtained: In 1879, Walter Flemming discovered cell mitosis and chromosomes, an achievement recognized as one of the 100 most important scientific achievements of all time.

20th Century

At the turn of the 19th/20th centuries Louis Pasteur invented pasteurization while Robert Koch discovered his famous or infamous postulates: the anthrax bacillus, the tuberculosis bacillus and the cholera vibrio.

UV and Phase: By 1900, the theoretic limit of resolution for visible light microscopes (2000 angstroms) had been reached. In 1904, Zeiss overcame this limitation with the introduction the first commercial UV microscope with resolution twice that of a visible light microscope. In 1930 Fritz Zernike discovered he could view unstained cells using the phase angle of rays. Spurned by Zeiss, his phase contrast innovation was not introduced until 1941 although he went on to win a Nobel Prize for his work in 1953.

Electron Microscopes: In 1931 Max Knoll and Ernst Ruska invented the first electron microscope that blasted past the optical limitations of the light. Physics dictates that light microscopes are limited by the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers.

Knoll and Ruska built a transmission electron microscope (TEM) - one that transmits a beam of electrons (as opposed to light) through the specimen. The subsequent interaction of the beam of electrons with the specimen is recorded and transformed into an image. Then, in 1942, Ruska improved on the TEM by building built the first scanning electron microscope (SEM) that transmits a beam of electrons across the specimen.

Ruska's principles still form the basis of modern electron microscopes - microscopes that can achieve magnification levels of up to 2 million times! The second major development for microscopes in the 20th century was the evolution of the mass market. Started in the 19th century when Leitz claimed to have exported 50,000 microscopes to the U.S., this trend accelerated in the 20th century. As a result, a large number of manufacturers sprang up to offer more competitively priced alternatives to established European companies such as Zeiss and Leitz.

China: China has become a major supplier of microscopes for everyday use and, with the evolution of their optical manufacturing capability, now supplies optical components to some of the major microscope brands. This market trend has had a beneficial effect on the price of microscopes, enabling the spread of microscopes beyond the realm of the research scientist to everyday commercial and individual use.

New light sources - halogen, fluorescent and LED have all improved or added a greater versatility of the light microscope, while the advent of boom stands have led to extensive commercial inspection applications that cannot be undertaken with a standard pedestal microscope base. The most recent innovation, however, has been the arrival of the digital microscope.

Digital Microscopes: Digital microscopes allow for live image transmission to a TV or computer screen and have helped revolutionize microphotography. Digital microscopes simply integrate a digital microscope camera on the trinocular port of a standard microscope. An alternative and more flexible solution is simply to place a digital microscope camera on a trinocular microscope!

Dino-Lite: One of the more original innovations in the 21st century has been Dino-Lite Digital microscopes. Dino-Lite are handheld digital microscopes, not much larger than a fat pen. They offer low power zoom capability with magnification up to 500x. They have had a marked impact on industrial inspection applications.


Aberration

Various aberrations influence the sharpness or quality of the image. Chromatic aberrations produce coloured fringes about the high-contrast regions of the image, because longer wavelengths of light (such as red) are brought to focus in a plane slightly farther from the lens than shorter wavelengths (such as blue). Spherical aberration produces an image in which the centre of the field of view is in focus when the periphery may not be and is a consequence of using lenses with spherical (rather than nonspherical, or aspheric) surfaces. Distortion produces curved images from straight lines in the object. The type and degree of distortion visible is intimately related to the possible spherical aberration in the magnifier and is usually most severe in high-powered lenses.


How a Scanning Electron Microscope Works

  • Source of electrons
  • Column down which electrons travel with electromagnetic lenses
  • Electron detector
  • Sample chamber
  • Computer and display to view the images

Electrons are produced at the top of the column, accelerated down and passed through a combination of lenses and apertures to produce a focused beam of electrons which hits the surface of the sample. The sample is mounted on a stage in the chamber area and, unless the microscope is designed to operate at low vacuums, both the column and the chamber are evacuated by a combination of pumps. The level of the vacuum will depend on the design of the microscope.

Schematic of a Scanning Electron Microscope

The position of the electron beam on the sample is controlled by scan coils situated above the objective lens. These coils allow the beam to be scanned over the surface of the sample. This beam rastering or scanning, as the name of the microscope suggests, enables information about a defined area on the sample to be collected. As a result of the electron-sample interaction, a number of signals are produced. These signals are then detected by appropriate detectors.


ELECTRON MICROSCOPE OBSERVATIONS ON THE SUBMICROSCOPIC ORGANIZATION OF THE RETINAL RODS

Eduardo De Robertis ELECTRON MICROSCOPE OBSERVATIONS ON THE SUBMICROSCOPIC ORGANIZATION OF THE RETINAL RODS . J Biophys and Biochem Cytol 25 May 1956 2 (3): 319–330. doi: https://doi.org/10.1083/jcb.2.3.319

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium.

The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments.

The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments.

In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber.

The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.


Microscopes

Olympus is a leading manufacturer of microscopes for life science and industry. With over 100 years of experience developing microscopes, we offer innovative optical solutions for many applications. Explore our microscopes for education, training, laboratories, and leading-edge research in the life science fields, such as pathology and cytology. Check out our latest laser scanning, super resolution, stereo, upright, inverted, and research macro zoom microscope systems by clicking the links below.


2.3: Instruments of Microscopy - Biology

Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera. This group of instruments includes not only multiple-lens (compound microscopes) designs with objectives and condensers, but also very simple single lens instruments that are often hand-held, such as a loupe or magnifying glass.

The microscope illustrated in Figure 1 is a simple compound microscope invented by British microscopist Robert Hooke sometime in the 1660s. This beautifully crafted microscope has an objective lens near the specimen and is focused by turning the body of the microscope to move the objective closer to or farther from the specimen. An eyepiece lens is inserted at the top of the microscope and, in many cases, there is an internal "field lens" within the barrel to increase the size of the viewfield. The microscope in Figure 1 is illuminated through the oil lamp and water-filled spherical reservoir, also illustrated in Figure 1. Light from the lamp is diffused when it passes through the reservoir and is then focused onto the specimen with a lens attached to the reservoir. This early microscope suffered from chromatic (and spherical) aberration, and all images viewed in white light contained "halos" that were either blue or red in color.

Since so many microscope users rely upon direct observation, it is important to understand the relationship between the microscope and the eye. Our eyes are capable of distinguishing color in the visible portion of the spectrum: from violet to blue to green to yellow to orange to red the eye cannot perceive ultraviolet or infrared rays. The eye also is able to sense differences in brightness or intensity ranging from black to white and all the gray shades in between. Thus, for an image to be seen by the eye, the image must be presented to the eye in colors of the visible spectrum and/or varying degrees of light intensity. The eye receptors of the retina used for sensing color are the cone cells the cells for distinguishing levels of intensity, not in color, are the rod cells. These cells are located on the retina at the back of the inside of the eye. The front of the eye (see Figure 2), including the iris, the curved cornea, and the lens are respectively the mechanisms for admitting light and focusing it on the retina.

For an image to be seen clearly, it must spread on the retina at a sufficient visual angle. Unless the light falls on non-adjacent rows of retinal cells (a function of magnification and the spreading of the image), we are unable to distinguish closely-lying details as being separate (resolved). Further, there must be sufficient contrast between adjacent details and/or the background to render the magnified, resolved image visible.

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Because of the limited ability of the eye's lens to change its shape, objects brought very close to the eye cannot have their images brought to focus on the retina. The accepted conventional viewing distance is 10 inches or 25 centimeters.

More than five hundred years ago, simple glass magnifiers were developed. These were convex lenses (thicker in the center than the periphery). The specimen or object could then be focused by use of the magnifier placed between the object and the eye. These "simple microscopes" could spread the image on the retina by magnification through increasing the visual angle on the retina.

The "simple microscope" or magnifying glass reached its highest state of perfection, in the 1600's, in the work of Anton von Leeuwenhoek who was able to see single-celled animals (which he called "animalcules") and even some larger bacteria with a simple microscope similar to the one illustrated in Figure 3. The image produced by such a magnifier, held close to the observer's eye, appears as if it were on the same side of the lens as the object itself. Such an image, seen as if it were ten inches from the eye, is known as a virtual image and cannot be captured on film.

Around the beginning of the 1600's, through work attributed to the Janssen brothers (see the microscope in Figure 4) in the Netherlands and Galileo in Italy, the compound microscope was developed. In its simplest form, it consisted of two convex lenses aligned in series: an object glass (objective) closer to the object or specimen and an eyepiece (ocular) closer to the observer's eye (with means of adjusting the position of the specimen and the microscope lenses). The compound microscope achieves a two-stage magnification. The objective projects a magnified image into the body tube of the microscope and the eyepiece further magnifies the image projected by the objective.

Compound microscopes developed during the seventeenth and eighteenth centuries were hampered by optical aberration (both chromatic and spherical), a flaw that is worsened by the use of multiple lenses. These microscopes were actually inferior to single lens microscopes of the period because of these artifacts. The images they produced were often blurred and had the colorful halos associated with chromatic aberrations that not only degrade image quality, but also hamper resolution. In the mid 1700's lens makers discovered that by combining two lenses made of glass with different color dispersions, much of the chromatic aberration could be reduced or eliminated. This discovery was first utilized in telescopes, which have much larger lenses than microscopes. It wasn't until the start of the 1800's that chromatically corrected lenses became commonplace in compound microscopes.

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The eighteenth and nineteenth centuries witnessed a great improvement in the mechanical and optical quality of compound microscopes. Advances in machine tools allowed more sophisticated parts to be fabricated and, by the mid 1800's, brass was the alloy of choice for the production of high-quality microscopes. A number of British and German microscope manufacturers flourished during this time period. Their microscopes varied widely in design and production quality, but the overall principles defining their optical properties remained relatively constant. The microscope illustrated in Figure 5 was manufactured by Hugh Powell and Peter Lealand about 1850. The tripod base provided a sturdy support for the microscope, which many people consider the most advanced of its period.

By the end of the nineteenth century, there was a high degree of competition among microscope manufacturers and the development and production costs of microscopes became an important factor. Brass, the material of choice for microscope manufacturers, is very expensive and it was a lengthy task to machine, polish, and lacquer microscope bodies and other parts machined from this metal. To cut expenses, microscope manufacturers first started to paint the exterior portion of the microscope body and stand, as well as the stage and other non-moving parts.

During the first quarter of the twentieth century, many microscope manufacturers had begun substituting cast iron for brass in microscope frames and stages. Iron was much cheaper and could be not be distinguished from brass when painted black. They also started to electroplate many of the critical brass components such as knobs, objective barrels, nosepieces, eyepieces, and mechanical stage assemblies (illustrated in Figure 6). These early twentieth century microscopes still subscribed to a common design motif. They were monocular with a substage mirror that was used with an external lamp to illuminate the specimen. A typical microscope of the period is the Zeiss Laboratory microscope pictured in Figure 6. This type of microscope is very functional and many are still in use today.

Modern microscopes far exceed the design specifications of those made prior to the mid 1900's. Glass formulations are vastly improved allowing greater correction for optical aberration than ever before, and synthetic anti-glare lens coatings are now very advanced. Integrated circuit technology has allowed manufacturers to produce "smart" microscopes that incorporate microprocessors into the microscope stand. Photomicrography in the late twentieth century is easier than ever before with auxiliary attachments that monitor light intensity, calculate exposure based on film speed, and automatically perform complicated tasks such as bracketing, multiple exposure, and time-lapse photography.

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The microscope illustrated in Figure 7 is an Olympus Provis AX70 research microscope. This microscope represents the latest state-of-the-art design that incorporates multiple illuminators (episcopic and diascopic), analyzers and polarizers, DIC prisms, fluorescence attachments, and phase contrast capabilities. The photomicrography system is the ultimate in sophistication and performance featuring spot measurement, automatic exposure control, and zoom magnification for flexible, easy framing. The Y-shaped frame is designed to be user-friendly by offering the maximum in operator comfort and ease of use.

The previous discussion addressed the basic concept of what a microscope is and touched upon an abbreviated history beginning in the seventeenth century and progressing through modern times. There are a number of additional topics that are of paramount importance towards gaining a complete understanding of microscopes and microscopy. These topics will be discussed in subsequent sections of the primer.

Practically everyone has, at one time or another, viewed the world through an optical microscope. For most people, this experience occurs during biology training in high school or college, although some scientific entrepreneurs have purchased their own microscopes either individually or as part of a science kit. Photography through the microscope, or more commonly, photomicrography, has long been a useful tool to scientists. For many years, the biological and medical sciences have relied heavily on microscopy to solve problems relating to the overall morphological features of specimens as well as a quantitative tool for recording specific optical features and data. In this respect, the optical microscope has proven useful in countless investigations into the mysteries of life.

Interactive Tutorial
Reflected Microscopy Light Pathways Explore the basic pathways of light through a reflected (episcopic) light microscope.

More recently, microscopy has enjoyed an explosive growth as a tool in the physical and materials sciences as well as the semiconductor industry, due to the need to observe surface features of new high-tech materials and integrated circuits. Microscopy is also becoming an important tool for forensic scientists who are constantly examining hairs, fibers, clothing, blood stains, bullets, and other items associated with crimes. Modern advances in fluorochrome stains and monoclonal antibody techniques have heralded an explosive growth in the use of fluorescence microscopy in both biomedical analysis and cell biology.

Interactive Tutorial
Fluorescence Microscopy Light Pathways Explore reflected light pathways and dichroic filtering in fluorescence microscopy.

The basic differences between biomedical and materials microscopy involves how the microscope projects light onto the sample. In classical biological microscopy, very thin specimens are prepared and the light is passed or transmitted through the sample, focused with the objective and then passed into the eyepieces of the microscope. For observing the surface of integrated circuits (that comprise the internal workings of modern computers) light passed through the objective and is then reflected from the surface of the sample and into the microscope objective. In scientific nomenclature, transmitted and reflected light microscopy are known as diascopic and episcopic illuminated microscopy, respectively. The photomicrographs in our photo galleries are derived from both transmitted and reflected optical microscopic scientific investigations.

One of the most serious problems in microscopy is the poor contrast produced when light is passed through very thin specimens or reflected from surfaces with a high degree of reflectivity. To circumvent this lack of contrast, various optical "tricks" have been perfected by scientists to increase contrast and to provide color variations in specimens. The assortment of techniques in the microscopists bag include: polarized light, phase contrast imaging, differential interference contrast, fluorescence illumination, darkfield illumination, Rheinberg illumination, Hoffman modulation contrast, and the use of various gelatin optical filters. A thorough discussion of these techniques is provided in the Specialized Microscopy Techniques section of this primer. References are provided in both classical bibliographic form and as website links in the front-end page of the microscopy primer. These should serve to provide more details of microscopy and photomicrography to interested readers as well as links to additional material on the World Wide Web.

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


2.3: Instruments of Microscopy - Biology

FORMS REQUIRED FOR THIS LAB

The models found in most schools, use compound lenses and light to magnify objects. The lenses bend or refract the light, which makes the object beneath them appear closer.

This microscope allows for binocular (two eyes) viewing of larger specimens. (The spinning microscope at the top of this page is a stereoscope)

Scanning Electron Microscope (SEM)

This microscope allows scientists to view a universe too small to be seen with a light microscope. SEMs dont use light waves they use electrons (negatively charged electrical particles) to magnify objects up to two million times.

Transmission Electron Microscope (TEM)

This microscope also uses electrons, but instead of scanning the surface (as with SEM's) electrons are passed through very thin specimens.

The microscope has been one of the key instruments used by the biologist for hundreds of years. A number of improvements were made to light microscopes during the first two or three centuries after their invention. Since the turn of the century, however, there have been no significant improvements. There have been continuous advances in the methods of preparing and analyzing specimens. There have also been modifications to the light microscope which permit new methods of analysis (phase contrast, fluorescence, confocal laser scanning, etc.) and the introduction of the electron microscope which can push microscopic analysis to the molecular level.

The main purpose of a microscope is to magnify and increase the visibility of a small object. The magnification of a lens is always engraved on it. You will be using a compound microscope which has a system of lenses. The total
magnification of the scope is a product of the magnifications of the objective lens and the eyepiece (or ocular lens). For example, using a low-power objective (magnification = 3.4X) and a standard eyepiece (magnification = 10X), the total magnification is 34X.

Resolution is an important attribute of a microscope. The limit of resolution of an optical system is the minimum distance by which two objects can be separated and still be perceived as distinct. Two points placed closer than this limit will be seen as one. Greater resolution allows one to see an object more sharply and to make out internal detail.

Adequate lighting will allow you to obtain the best resolution possible. Illumination must be adjusted for each objective every time a change is made. The adjustments which will affect illumination on your microscope involve changing the iris diaphragm. The iris diaphragm is used to match the aperture (opening) to that of the objective. It should not be used to control the intensity of illumination. With some unstained or transparent specimens, it may be necessary to close the iris slightly to improve contrast. This is always done at the expense of resolution. Too much light through the diaphragm will wash out the specimen you are viewing similar to viewing an object with a bright light in the background.

SKETCH 1
**Sketch a compound light microscope and label the following: E yepiece, Objective, Stage,
Fine Adjustment, Coarse Adjustment, Diaphragm

Link below to quiz yourself on the parts of a compound light microscope. You do not need to submit the answers for the quiz with your lab report.

An alternative link to the Microcope video is given below.


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Figure 1.10
Alveolar Cell Carcinoma

In the United States, lung cancer is the leading cause of cancer-related death among both men and women. The popularity of smoking tobacco throughout the twentieth century is usually considered accountable for the prevalence of the disease, cigarettes having been linked to about 90 percent of lung cancer cases in men and 80 percent in women. Yet, alveolar cell carcinoma appears to have no relationship to smoking. Also known as bronchoalveolar carcinoma, alveolar cell carcinoma instead appears to be most likely to develop in individuals whose lungs have been scarred by other diseases, such as scleroderma, tuberculosis, or fibrosis. The progression of this variety of carcinoma can be very slow, and patients with the disease often have a better prognosis than people with other kinds of lung cancer. View the microscopic image above of alvolar cell carcinoma. The darker purple organelles you view in the individual cells are the nuclei of the alveolar cells in the lungs. Normal nuclei should be round or oblong in shape. Notice that many of the nuclei are misshapen (triangular or cuboidal). These misshapened nuclei are indicative of abnormal carcinoma cells. Click on the link below to view the same image above with many of the cancer cells circled in green.

I) Bacteria

Observe the following image which illustrates the three different types of bacteria. Use the links given below to view microscopic slides of each of the three shapes of bacteria.

SKETCH 2
**Identify the different shapes of bacteria in your sketch as: Baccilus, Cocci, Spirillum

Baccillus (Rod shaped)

Cocci (Spherical shaped)

Spirillum (Spiral shaped)

II) Adipose Tissue

The image to the right is a 100X microscopic view of a cross section of adipose tissue. The large white structures are the adipose (fat) cells. The smaller, dark red structures are the nuclei of the individual cells.

SKETCH 3
**Sketch just a few of the cells in the image to your right. The dark red objects you can see are the nuclei of the cells

Video of a 100X microscopic view of a drop of pond water. Click on the arrow to view the video.

The variety of organisms you see here would be typical of most freshwater ponds. The small spherical organisms seen floating about are bacteria. The large and small, fast moving, green organisms that are darting about are paramecium feeding on bacteria. The hat shaped organisms are vorticella again feeding on bacteria. The darker greenish brown masses are bacterial colonies and algae. The green rectangular organisms are green algae. The long, thin strands scattered throughout the sample are blue-green algae.

An alternative link can be found below :

Some Interesting Movies Using Light Microscopy

Click on the links below to see a Water Bear moving under a microscopic power of 250X

Water Bear 1

Water Bear 2

An amoeba engulfs a food item in a process called phagocytosis at a magnification of 400x

Pseudopodia, or false feet, extend and retract as this amoeba moves across the microscope field at a magnification of 100x

An up close and personal look at the internal workings of a paramecium at a magnification of 400x

A group of swimming paramecia (the smaller organisms), looking more like a bunch of bumper cars as they collide and rebound off of one another

Using the microscope to view the differences between normal cells and cancer cells

1) What is the function of the diaphragm?
2) Describe the advantage of having a microscope of highest resolution.
3) Calculate the magnification of the lens system of the following:
a) Ocular-10X Objective-10X
b) Ocular-10X Objective-43X
c) Ocular-10X Objective-1X
d) Ocular-10X Objective-2X
4) What is the most important attribute of a microscope?

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions

**Go to the following site to link to a virtual light microscope. At the virtual microscope site you will need to perform the tutorial so that you learn how to use the microscope. Click on the GETTING STARTED link on the upper left side. After learning how to use the microscope and viewing the speciments, answer the questions given below on Virtual Light Microscopy.


VIRTUAL LIGHT MICROSCOPE

A) Perform the tutorial (Getting Started) so that you learn how to use the microscope
B)
There are four slides to view. Choose to view the cheek smear slide. You may view the others if you wish however the questions will pertain to only the cheek smear slide.
C) You will be using the 10X, 40X and 100X objective lens powers
D) Use the focus and illumination slide bars to see the cheek cells better.
E) Answer the Virtual Light Microscopy Questions provided below.

You can use the page link below to access a labeled image of the microscope

1) How many individual cells can you count at the following objective powers of magnification?
a) 10X
b) 40X
c) 100X
2)
If the eyepiece has a 10X power, what is the total magnification when you observe cells at objective power of 40X?
3)
At what power are you able to discern the nucleus of the cells? (The nucleus is the large, darker organelle located near the center of the cell)
4)
Describe what happens if there is too much illumination.

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions

Please Find Below Links to Other Virtual Light Microscopy Sites

Two types of electron microscopes have been developed over the past half century: the T ransmission E lectron M icroscope (TEM) and the S canning E lectron M icroscope (SEM) . These instruments contain magnetic lenses that focus a beam of electrons on the specimen. Electrons used in this fashion generate a wavelength that may be 100,000 times shorter than that of visible light. As a result, electron microscopes have resolving powers as much as 400 times that of light microscopes and 200,000 times that of the human eye.

The TEM bombards a thin specimen with electrons. Depending on their composition, the components of the specimen either transmit, absorb or deflect the electrons. The image produced on a photographic plate is a visual translation of this interaction of electrons with the specimen. The transmission electron microscope gave scientists their first look at the world of viruses, invisible by light microscopy, and today permits us to see molecules and atoms.

The SEM is quite different from the TEM . It is designed to generate three-dimensional images of surface detail. This microscope moves an electron beam back and forth over the surface of a metal-coated specimen causing the emission of secondary electrons from the specimen. The secondary electrons produce the stunning images characteristic of scanning electron microscopy.

Video of how an electron microscope works. Click on the arrow to view the video.

An alternative link can be found below :


Figure 1.16
Transmission Electron Micrograph
of Polio Virus

Figure 1.17
Transmission Electron Micrograph
of Ebola virus

Video of numerous scanning electron microscopic views of cells from the human body. Click on the arrow to view the video.

An alternative link can be found below :

To observe (resolve) objects smaller than 0.2 m requires the utilization of Electron Microscopy (EM). Rather than using visible light, electron microscopes focus a beam of electrons on a very thin section of biological material that has been chemically preserved (fixed) and embedded in plastic. Electrons have a much shorter wavelength than the photons of visible light used in LM. Since resolving power is inversely related to wavelength, modern electron microscopes can resolve objects of approximately 0.2 m. It is this tremendous increase in resolution that has allowed biologists to discern the precise details of cell structure. Although a powerful tool, only chemically preserved cells can be observed with EM. The routine observation of living cells by electron microscopes is a goal yet to be achieved.

The type of electron microscopy described above is generally referred to as Transmission Electron Microscopy (TEM). In TEM, the beam of electrons passes directly through the sample except where the electrons are deflected by atoms of heavy metals (lead and/or uranium) that have been used to "stain" the specimen the transmitted electrons are focused onto photographic film where the image is visualized and recorded.

A variation on this approach is Scanning Electron Microscopy (SEM). In SEM, the electron beam scans the surface of a sample that has been coated with a thin layer of gold. The beam of electrons excites the atoms of the sample causing them to eject electrons which are collected and converted into an image that is displayed on a monitor. The image that is produced has a great depth of field and thus appears to be three dimensional. SEM is used to reveal the surface details of various types of cells.

**Go to the following site to experiment with a virtual scanning electron microscope. Answer the questions on Virtual Electron Microscopy given below.
VIRTUAL ELECTRON MICROSCOPE

A) There are three specimens to view shown on the left side
B) You can use the MAGNIFY button on the machine to zoom in on your specimen
C) Answer the questions on your experiences at this site below.

1) How many stem cells can you count?
2) Which of the cells are larger the stem cells or the T-cell
3) Which cell is the largest?
4) Describe the shape of the Red Blood cell
5) What do you think the pinkish strands are around the nerve fibers
6) Does the electron microscope allow a higher degree of magnification than the light microscope? Why?

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions

A Link to Another Virtual Electron Microscope

Something Interesting About Measurements

View the Milky Way at 10 million light years from the Earth. Then move through space towards the Earth in successive orders of magnitude until you reach a tall oak tree just outside the buildings of the National High Magnetic Field Laboratory in Tallahassee, Florida. After that, begin to move from the actual size of a leaf into a microscopic world that reveals leaf cell walls, the cell nucleus, chromatin, DNA and finally, into the subatomic universe of electrons and protons.

Measurements Movie

Try this great interactive measurements animation. Slide the slider at the bottom of the interaction to view smaller or larger items.

How big are bacteria and virus?

Use this site to view the relative size of things like amoeba, skin cells, chromosomes, bacteria, etc. Use the sliding bar at the bottom of the page to zoom in to smaller objects.

Scale


Watch the video: Lab Exercise 2: Microscopes and Cell Shapes (May 2022).


Figure 1.18
Scanning electron micrograph of
HIV grown in cultured lymphocytes


Figure 1.19
Scanning electron micrograph of
Treponema pallidum
(The causative agent of syphilis)