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1.3.3: Instruments of Microscopy - Biology

1.3.3: Instruments of Microscopy - Biology


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Learning Objectives

  • 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} imes ext{objective magnificationocular magnification}; imes; ext{objective magnification} onumber]

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

[(40×)(10×)=400× onumber]

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.

MICROSCOPE MAINTENANCE: BEST PRACTICES

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}))

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})).

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/C.W. Hubbard)

Exercise (PageIndex{1})

Identify the key differences between brightfield and darkfield microscopy.

Clinical Focus: Part 2

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})).

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.

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})).

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. (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})).

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})).

(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/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})).

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 electron microscope was invented in 1931 by Max Knoll and Ernst Ruska at the Berlin Technische Hochschule, but it was not commercially available until 1938.

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})).

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

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})).

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})).

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.

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).

Footnotes

  1. 1 “JEM-ARM200F Transmission Electron Microscope,” JEOL USA Inc, www.jeolusa.com/PRODUCTS/Tran...specifications. Accessed 8/28/2015.

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)


5 Important Types of Microscopes used in Biology (With Diagram)

Some of the most important types of microscopes that used in biology are as follows:

1. Simple microscope 2. Compound microscope 3. Electron microscopes 4. Phase-Contrast microscope 5. Interference microscope.

The simple dissection microscope to advanced electron microscopes finds application in studies of living organisms.

Microscope as the name suggests are instruments that help to enlarge minute (micro = very small) organisms or their parts. A microscope not only presents a magnified view of the object but also ‘resolves’ it better.

Resolution is the feature which makes it possible to differentiate between two points present close together in the objects being viewed. The first microscope was constructed by Anton Van Leeuwenhoek (1632-1723). This, microscope consisted of a single biconvex lens fitted in a small window of a “board” and the object was viewed through it. This was a simple microscope.

After this compound microscope, were developed using combinations of two lenses. Improvements continued, newer and newer’ microscopes were designed and are still being improved.

Different types of microscopes being used in biological studies are the following:

It is the ability of a microscope to show two closely lying points as two distinct points.

It is the ratio of the size of the image to that of the object:

4. Phase-Contrast microscope

5. Interference microscope

1. Simple Microscopes:

Simple/ Dissecting Microscope:

As shown in the figure 6.1, dissecting microscope consists of a biconvex lens which is moved up and down by an adjustment screw to bring the object in sharp focus. The object is placed on the platform and light is focused with the help of a concave mirror fitted below.

In simple microscope, convex lens of short focal length is used to see magnified image of a small object. The object is placed between the optical centre and the focus of a convex lens, its image is virtual, erect and magnified and on the same side as the object. The position of the object is so adjusted that the image is formed at the least distance of distinct vision (D).

Magnifying power (M) of a simple microscope is the ratio of the angle subtended by the image at the eye to the angle subtended by the object seen directly, when both lie at the least distance of distinct vision or the near point.

Where D is the least distance of distinct vision and f is the focal length of the lens.

2. A Compound Microscope:

A compound microscope consists of two set of convex lenses. A lens of short aperture and short focal length facing the object is called objective. Another set of lens of relatively moderate focal length and large aperture facing the eye is called the eye piece. The objective and the eye piece are placed coaxially at the two end of a tube (Fig. 6.2).

The object is placed between the centre of curvature and focus of the objective – it forms real, inverted and magnified image on the other side of the objective. This image acts as an object for the eye piece which then acts as a simple microscope to produce virtual, erect and magnified image.

Magnifying power (M) of a compound microscope will be

Where f0and fe are focal length of objective and eye piece respectively, L is the length a the microscope tube and D is the least distance of distinct vision.

3. The Electron Microscope:

The organelles of the cell became known after the electron microscope was invented. The electron microscope was developed in 1932 by M. Knoll and Ruska in Germany. It consist of a source of supplying, a beam of electron of uniform velocity, a condenser lens for concentrating the electron on the specimen, a specimen stage for displacing the specimen which transmits the electron beam, an objective lens, a projector lens and a fluorescent screen on which final image is observed (fig. 6.3).

For permanent record of the image, the fluorescent screen is replaced by photographic film. This microscope utilizes a stream of high speed electrons which are deflected by an electromagnetic field in the same way as a beam of light is reflected when it crosses a glass lens. There are two types of electron microscope.

(a) Transmission electron microscope (TEM):

This is used to observe fine structure of cells. Ultra thin sections of the object are prepared and they are stained with a heavy metal (gold or palladium) to make certain part dense, and inserted in the vacuum chamber of the microscope. A 100, 00 volt electron beam is focused on the section and manipulated prepared from the image may be enlarged with enough resolution to achieve a total magnification of over 20 million times.

(b) Scanning electron microscope (SEM):

It is used to study the surfaces of the cell and organisms. In this microscope, the image is formed by electrons reflected back from the object. The image formed by this microscope has a remarkable three dimensional appearance. Typically magnification of scanning electron microscope is around 20,000 times.

4. Phase-Contrast microscope:

This is used to study the behavior of living cells, observe the nuclear and cytoplasmic changes taking place during mitosis and the effect of different chemicals inside the living cells. By using the phase-contrast microscope, an image of strong contrast of the object is obtained (fig. 6.4).

It is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, fibers, glass fragments, and sub-cellular particles (including nuclei and other organelles). In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast.

One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without previously being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail.

5. Interference microscope:

Interference microscope is used for quantitative studies of macromolecules of the cell components, for example it is used for determination of lipid, nucleic acids and protein contents of the cell. Interferometry is a traditional technique in which a pattern of bright and dark lines (fringes) result from an optical path difference between a reference and a sample beam.

The incoming light is split inside an interferometer, one beam going to an internal reference surface and the other to the sample. After reflection, the beams recombine inside the interferometer, undergoing constructive and destructive interference and producing the light and dark fringe pattern.

A precision translation stage and a CCD camera together generate a 3D interferogram of the object that is stored in the computer memory. This 3D interferogram of the object is then transformed by frequency domain analysis into a quantitative 3D image providing surface structure analysis (fig. 6.5).


Light Microscopes

To give you a sense of cell size, a typical human red blood cell is about eight millionths of a meter or eight micrometers (abbreviated as eight μm) in diameter the head of a pin of is about two thousandths of a meter (two mm) in diameter. That means about 250 red blood cells could fit on the head of a pin.

Most student microscopes are classified as light microscopes (Figure 1a). Visible light passes and is bent through the lens system to enable the user to see the specimen. Light microscopes are advantageous for viewing living organisms, but since individual cells are generally transparent, their components are not distinguishable unless they are colored with special stains. Staining, however, usually kills the cells.

Light microscopes commonly used in the undergraduate college laboratory magnify up to approximately 400 times. Two parameters that are important in microscopy are magnification and resolving power. Magnification is the process of enlarging an object in appearance. Resolving power is the ability of a microscope to distinguish two adjacent structures as separate: the higher the resolution, the better the clarity and detail of the image. When oil immersion lenses are used for the study of small objects, magnification is usually increased to 1,000 times. In order to gain a better understanding of cellular structure and function, scientists typically use electron microscopes.

Figure 1. (a) Most light microscopes used in a college biology lab can magnify cells up to approximately 400 times and have a resolution of about 200 nanometers. (b) Electron microscopes provide a much higher magnification, 100,000x, and a have a resolution of 50 picometers. (credit a: modification of work by “GcG”/Wikimedia Commons credit b: modification of work by Evan Bench)


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.


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.


3D Correlative Microscopy Using Thresholding

Researchers at the Institut de Génétique et de Biologie Moléculaire et Cellulaire in Illkirch, France have developed a new method for correlating live-cell imaging with electron microscopy (EM) on cryofixed samples. According to Yannick Schwab, one of the research team&rsquos members, the group&rsquos goal was to develop a way to correlate light microscopy data acquired from living cells with ultrastructural analysis of the same objects.

Figure: COS-1 cells transfected with GFP-tagged BIN1 were imaged by confocal microscopy, fixed by high pressure freezing, processed for 60 nm thick serial sections, and labeled with anti-GFP antibody and gold particles. (A) Representative image (z projection of 3 confocal sections) of the fluorescent B1N1 tubules radiating from/to the perinuclear region. (B) One serial thin section showing the transmission-EM image of the same cell. (C) At higher magnification, the immunostaining is visible over various perinuclear structures (highlighted in yellow). Unstained organelles such as mitochondria, Golgi complex, and endosomal vesicles are shown in white. (D) Rendering the entire stack of serial sections allows the immunogold labeling to be projected onto the representative transmission-EM picture, showing a good correlation with the dense perinuclear GFP signal recorded on living cells. (E and F) The distribution of the anti GFP immunogold staining in the volume of the perinuclear region. (G&ndashJ) The full z-stack recording was processed in Imaris to reconstruct a 3D model (green) of the GFP fluorescence. The region analyzed by EM (yellow) shows a good correlation with the gold labeling in D-F. (K&ndashM) Correlation analysis of other z planes showed localization of GFP-BIN1 at plasma membrane invaginations. Image reprinted from PLoS One.

To accomplish this, the researchers pre-patterned a reference grid on aclar discs, which their cells were then cultured on. These discs are compatible with cryofixation and resin embedding. Moreover, the co-ordinates on the grid are conserved throughout preparation and EM imaging. The advantage of using this method is that it is compatible with various EM techniques, high-resolution live recording of dynamic phenomena, and fast high-pressure freezing.

In the initial phase of the experiment, the group acquired live-cell imaging data using either a confocal or epifluorescence microscope. Following this, the cells were subjected to either chemical fixation for scanning EM or high pressure freezing for electron tomography (or serial immuno-EM). Lastly, the data acquired from both microscope modalities were arranged to be analyzed and correlated in the Imaris 3D/4D image analysis software.

Using a specific 3D intensity thresholding function, unique to the Imaris software, the researchers were able to select specific objects in the data based on their intensity profiles. Once all of the objects in the data set were identified, Schwab&rsquos group filtered away all of the smaller, unwanted objects in order to isolate only the structures of interest. In combination, the specific thresholding and 3D filtering applications in Imaris allowed the precise determination of the shape and location of specific membrane tubules that form in the perinuclear region of cells transfected with GFP-tagged BIN1.

In the next phase of the experiment, the data extracted from the analysis in Imaris was used to select a set of thin sections to study at the EM level. Using these sections, the researchers tried to elucidate an immuno-EM correlation. From this final analysis, Schwab&rsquos group ascertained that the anti-GFP immunogold distribution across several serial EM sections matched the fluorescent pattern highlighted in Imaris (see figure)

&ldquoWe managed to get ultrastructural snapshots of dynamic events or to study the 3-D organization of selected subcellular compartments, both with confocal microscopy and with serial or electron tomography,&rdquo Schwab said.

Author: Dr. Coralie Spiegelhalter and colleagues,Institut de Génétique et de Biologie Moléculaire and other


Amira for Cell Biology

Understanding living cells and their processes requires analysis of data from various imaging systems and modalities that all use different file formats. Each experimental setup can represent a unique challenge to process the data. Multi-scale dynamic processes require tracking of various sized objects, from diffraction-limited particles to entire cells. There can be a few dozen to tens of thousands objects in each data set. In addition, intracellular processes are supported by sub-cellular structures that also need to be identified and quantified.
Thermo Scientific Amira Software provides a comprehensive array of tools for the flexible and accurate analysis of time series data of cellular processes. It enables researchers to perform dedicated segmentation workflows on their intra- and intercellular images and apply powerful automated object tracking solution.

To better understand embryonic development and how congenital disorders occur, scientists need to follow individual cells during the development cycle of an embryo. Using powerful, high-speed light-sheet microscopy, researchers are able to image these cells at sufficient temporal and spatial resolution.

With Amira Software, researchers are now able to deploy custom segmentation algorithms to segment each individual cell in a time series of images. These thousands of segmented cells can then be tracked using Amira Software's automated powerful tracking solution (powered by u-track 3D , under submission for peer-review from the Danuser Lab). With Amira automatic selection of the appropriate motion model, event detection, and gap closing, researchers can track thousands of fast-moving cells with high precision.

From these tracks, Amira Software enables scientists to extract quantitative information such as shape size and intensity parameters or measurements related to motility and diffusion. Researchers can also analyze the track across multiple cell generations to obtain cell lineage information.


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.

Figure 10. A transmission electron microscope (TEM).

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 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 11).

Figure 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 12).

Figure 12. These schematic illustrations compare the components of transmission electron microscopes and scanning electron microscopes.

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 13). Some EMs can magnify an image up to 2,000,000×. [1]

Figure 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)

Think about It

  • What are some advantages and disadvantages of electron microscopy, as opposed to light microscopy, for examining microbiological specimens?
  • 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 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.

Figure 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)

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 15).

Figure 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)


Choosing a Microscope

And as far as buying a microscope, don't spend exhaustive amounts of time in the research process. Better understand and increasingly enjoy this growing field of study by letting MicroscopeMaster help you to make the right choice!

Are you wanting to find the best compound or stereo microscope to suit your needs or simply learn more about microscopy? Cheap microscopes without sacrificing quality are available and featured here.

Details in specimens that can be magnified today certainly reveal an exciting depth to our world.

At whichever level you may be at, a student or professional, beginner to advanced, broadening your knowledge will add to your discovery of this fascinating field.


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.


Watch the video: basic histopathology 3 Other types of microscope chapter 1 (July 2022).


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