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2.4.3: Interference Microscopy - Biology

2.4.3: Interference Microscopy - Biology


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LEARNING OBJECTIVES

  • Describe the principles and different types of interference microscopy

Stereo Light Source

Interference microscopy uses a prism to split light into two slightly diverging beams that then pass through the specimen. It is thus based on measuring the differences in refractive index upon recombining the two beams. Interference occurs when a light beam is retarded or advanced relative to the other.

There are three types of interference microscopy: classical, differential contrast, and fluorescence contrast. Since its introduction in the late 1960s differential interference contrast microscopy (DIC) has been popular in biomedical research because it produces high-resolution images of fine structures by enhancing the contrasted interfaces. The image produced is of a thin optical section and appears three-dimensional, with a shadow around it. This creates a contrast across the specimen that is bright on one side and darker on the other.

The Interference Microscope

The microscope is a bright field light microscope with the addition of the following elements: a polarizer between the light source and the condenser, a DIC beam-splitting prism, a DIC beam-combining prism, and an analyzer. Manipulating the prism changes the beam separation, which alters the contrast of the image. When the two beams pass through the same material across the specimen they produce no interference. When the two beams pass through different material across the specimen such as on the edges, they produce alteration when combined.

Fluorescence differential interference contrast (FLIC) microscopy was developed by combining fluorescence microscopy with DIC to minimize the effects of photobleaching on fluorochromes bound to the stained specimen. The same microscope is equipped to simulataneously image a specimen using DIC and fluorescence illumination.

Key Points

  • Interference microscopy is superior to phase-contrast microscopy in its ability to eliminate halos and extra light.
  • In differential interference contrast microscopy (DIC), the optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the thickness traversed by a light beam between two points on the optical path.
  • Images produced by DIC have a distinctive shadow-cast appearance.

Key Terms

  • photobleaching: The destruction of a photochemical fluorescence by high-intensity light
  • fluorochrome: Any of various fluorescent dyes used to stain biological material before microscopic examination

2.4.3: Interference Microscopy - Biology


Of all the techniques used in biology microscopy is probably the most important. The vast majority of living organisms are too small to be seen in any detail with the human eye, and cells and their organelles can only be seen with the aid of a microscope. Cells were first seen in 1665 by Robert Hooke (who named them after monks' cells in a monastery), and were studied in more detail by Leeuwehoek using a primitive microscope.

Units of measurement

Magnification and Resolution [back to top]

By using more lenses microscopes can magnify by a larger amount, but this doesn't always mean that more detail can be seen. The amount of detail depends on the resolving power of a microscope, which is the smallest separation at which two separate objects can be distinguished (or resolved).

The resolving power of a microscope is ultimately limited by the wavelength of light (400-600nm for visible light). To improve the resolving power a shorter wavelength of light is needed, and sometimes microscopes have blue filters for this purpose (because blue has the shortest wavelength of visible light).

Magnification is how much bigger a sample appears to be under the microscope than it is in real life.

Overall magnification = Objective lens x Eyepiece lens

Resolution is the ability to distinguish between two points on an image i.e. the amount of detail

  • The resolution of an image is limited by the wavelength of radiation used to view the sample.
  • This is because when objects in the specimen are much smaller than the wavelength of the radiation being used, they do not interrupt the waves, and so are not detected.
  • The wavelength of light is much larger than the wavelength of electrons, so the resolution of the light microscope is a lot lower.
  • Using a microscope with a more powerful magnification will not increase this resolution any further. It will increase the size of the image, but objects closer than 200nm will still only be seen as one point.

2.3 Instruments of Microscopy

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 2.12 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:

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

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 with chromophores (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 2.13). A variety of oils can be used for different types of light.

Micro Connections

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

Link to Learning

Visit the online resource linked below for simulations and demonstrations involving the use of microscopes. Keep in mind that execution of specific techniques and procedures can vary depending on the specific instrument you are using. Thus, it is important to learn and practice with an actual microscope in a laboratory setting under expert supervision.

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 2.14).

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 2.15).

Check Your Understanding

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?

Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box.

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 2.16).

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 2.17).

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 2.18)

Check Your Understanding

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 2.19).

Check Your Understanding

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 2.20).

Link to Learning

Explore a rotating three-dimensional view of a biofilm as observed under a confocal microscope. After navigating to the webpage, click the “play” button to launch the video.

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.

Check Your Understanding

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 2.21). 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 2.22).

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 2.23). 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 2.24). Some EMs can magnify an image up to 2,000,000⨯. 1

Check Your Understanding

  • 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?

Micro Connections

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 2.25). 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 2.26).

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 2.27).

Figure 2.28, Figure 2.29, and Figure 2.30 summarize the microscopy techniques for light microscopes, electron microscopes, and scanning probe microscopes, respectively.

Check Your Understanding

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

Footnotes

    “JEM-ARM200F Transmission Electron Microscope,” JEOL USA Inc, http://www.jeolusa.com/PRODUCTS/TransmissionElectronMicroscopes%28TEM%29/200kV/JEM-ARM200F/tabid/663/Default.aspx#195028-specifications. Accessed 8/28/2015.

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    • Authors: Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster
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    Self-Assembly Processes at Interfaces

    3.3.6 Scanning Near-Field Optical Microscopies and Spectroscopy

    SNOM (Chapter 3.2.4) has been used to irradiate protein films on silicon and to obtain their mid-IR spectra with a spatial resolution of about 10 nm obtained through the near-field optics. First the IR spectra of individual ferritin molecules and those of the tobacco mosaic virus (TMV) were compared and found to be identical to the IR spectrum obtained in the far field under grazing incidence irradiation. The technique was then used to differentiate insulin aggregates rich in β-sheets from TMV rich in α-helixes mixed together. The spatial resolution afforded by the technique allowed to distinguish TMV from insulin, which both appeared in the form of filaments in the SNOM image [182] .


    Microscope: Types of Microscope

    The following points highlight the thirteen main types of microscopes. Some of the types are: Simple Microscope 2. Compound Microscope 3. Research Microscope 4. Binocular Dissection Microscope 5. Phase Contrast Microscope 6. Interference Microscope 7. Differential Interference Contrast Micro­scope 8. Fluorescent Microscope 9. Electron Microscope 10. Transmission Electron Microscope and Others.

    Type # 1. Simple Microscope:

    1. A simple microscope is a convex lens of short focal length.

    2. It is used to form a virtual image of an object placed just inside its principal focus.

    3. It is made up of a single convex lens or a combination of lenses which functions as a convex lens.

    4. The convex lens magnifies the object and also helps to produce a magnified image of a near object which appears to be at the distance of distinct vision.

    5. Under simple microscope the enlarged image (Fig. 143) of the object is formed on the retina of the eye of the viewer.

    6. A simple lens can magnify an object only three times OX). For getting a magnification of more than 3X, a combination of several lenses is used. Such a combination of several lenses (called elements) functions as a single convex lens, and a magnification of about 20X can be obtained.

    7. Improved simple microscopes, used by the biologists during field work, may magnify an object even up to 100 times. In such microscopes, multi­element lenses are used. It consists of a combination of a double concave lens of crown glass fitted between two double convex lenses of flint glass. This is called an aplanarlens or achromatic triplet. Three of such aplanar lenses are cemented together and function as one lens.

    A dissecting microscope (Fig.144) is an example of a simple microscope used either for dissecting the material or for viewing the magnified image of the material. It consists of only one lens unit. This lens unit may even be an ordinary magnifying glass.

    It is used either for dissecting the material or for less magnifications, i.e., only 6X, 12X or rarely 20X. It is mainly used for embryo separation, taxonomic studies, etc. It has a basal foot and a limb. The ‘stage’, made up of a simple glass plate, is attached to the limb.

    For the light adjustment purposes, a mirror is attached to the limb under the stage. Mirror can be moved vertically with the help of an adjustment screw. At the tip of the limb is present a folded arm, on which a lens of definite magnification (6X, 12X, etc.) is fitted. Folded arm is moved to keep the lens in the desired position on the stage.

    The material to be viewed or dissected under a dissecting microscope is placed on the stage. The eye is placed close to the lens. Folded arm is tilted to bring the lens over the material. Light is adjusted by the movement of the mirror. Focusing is done with the help of the adjustment screw of this microscope.

    Type # 2. Compound Microscope:

    A compound microscope (Fig.145) comprises either two (objectives and eyepiece) or three (condenser, objectives and eyepiece or ocular) kinds of lenses. The condenser, located above the mirror and below the stage, collects and focuses the light rays into the plane of the object.

    The objectives (10X, 40X, 100X) are mounted on a revolving nosepiece. Each objective consists of a set of elements fused together to work as a single lens. The objectives collect light rays from the object and form a magnified real image at some distance above them (Fig. 146).

    The eyepieces or oculars are located at the top of the body tube. The eyepiece usually have 5X, 10X or 15X lenses. The eyepiece magnifies the image formed by the objective (Fig. 146). A microscope with one ocular or eyepiece is called monocular microscope while the one with two oculars is called binocular microscope.

    1. The instrument is so named because it consists of two or more lens systems (Fig. 145).

    2. At the top is present the ocular lens. It can be turned around or may be removed. At the top of ocular lens is written 5X or 10X signifying the 5 times or 10 times magnification, respectively.

    3. Just below the ocular is a body tube, the bottom end of which contains a circular piece called nose piece. It contains three lenses called objective lenses. Nose piece can be rotated to change the position of objectives.

    4. The flat platform present below the objectives is called stage.

    5. On the arm of the microscope are present two knobs named coarse adjustment knob and fine adjustment knob.

    6. Out of the three objectives, the shortest is low power objective. It has the largest lens but its magnifying power is least of the objective lenses. On the objective may also be written 10X as on ocular lens. It means if a 10X ocular lens is used the magnification is 10 X 10 – 100 times.

    7. The other objective is high power objective. Its magnification is equal to the number written on it multiplied by the power of ocular i.e. 5X or 10X (objective x ocular).

    8. The third objective is oil immersion. Generally, it contains a black band around the lower end. A drop of oil is used on the slide at the time of studying with the oil immersion objective. Its magnification can be estimated as ocular x objective.

    The use of oil is essential in order to keep the light rays properly aligned with the small objective.

    9. Just below the stage is the condenser. Its function is to gather light from the mirror and direct it to the objective lens. Condenser may be lowered or raised by a knob present on one side of the microscope beneath the stage.

    10. Condenser contains a shutter called iris diaphragm.

    11. Just below the condenser is present a mirror having its one surface flat and other concave? Use the concave surface in the day light. Flat surface of the mirror is used when electric lamp is used.

    1. Clean the ocular and objective lenses with lens paper, and do not remove them.

    2. While studying an object, learn to keep one hand on the fine focus knob and focus continually up and down.

    3. While studying any kind of preparation, do not tilt the microscope.

    4. Leave the low power objective in place after finishing all the observations.

    5. To examine an object, always use the low power first and then the other objectives.

    6. Never allow an objective lens to strike either the stage or a slide while focusing.

    7. Use always the fine adjustment with high power objective.

    8. All wet-mount preparations should be pre-covered by a cover slip.

    9. Avoid the habit to remove the parts of the microscope.

    10. Do not use oil immersion objective without oil.

    11. Diaphragm should be wide open while using oil immersion objective.

    Type # 3. Research Microscope:

    1. It is a compound microscope having very fine quality lenses and some additional facilities such as oil immersion lens, built-in illumination, binocular viewing and attachment for photographic camera.

    2. The 100X objective is called an oil immersion lens. It can be used only by introducing a drop of an immersion oil between the objective and the cover-glass. If the oil is not used, the Image of the object will appear blurred. The specific gravity of immersion oil is equivalent to glass.

    3. In the built-in illumination facility, the mirror of the microscope is replaced by an electric bulb housed in a chamber fitted with a diaphragm.

    4. Binocular viewing means instead of one eye the researcher can use both the eyes together for viewing. For this facility, this microscope is fitted with two eyepieces instead of one.

    5. Microscopes with attachment for photographic camera are provided with trinocular tubes. Of these three tubes, two are provided with two oculars for binocular viewing while the third tube accommodates a camera for taking photographs.

    6. By using 15X ocular and 100X objective, a maximum magnification of 1500 times may be obtained under a research microscope.

    Type # 4. Binocular Dissection Microscope:

    1. In this microscope two microscopic systems are mounted alongside (Fig. 147).

    2. The two sets of eyepieces and objectives of this microscope produce two images which are erected by two prisms present inside the body.

    3. Sufficient space is present between the objective and the stage. This helps in the easy dissection and proper study of the material.

    4. This microscope is used for doing fine dissections of the specimens and to study their gross mounts because a three-dimensional picture is available due to binocular viewing facility.

    5. The maximum magnification obtained by this microscope is 150X.

    Type # 5. Phase Contrast Microscope:

    1. It was discovered by Professor Frits Zernike of Netherlands. Its commercial production was first started in Germany in 1942. For the recognition of his discovery of phase contrast, Zernike was awarded Noble Prize for Physics in 1953.

    2. Only living and unstained cells are studied under phase contrast microscope. It is so because the living cells are not usually coloured (i.e. they are pure phase objects), but the light transmitted by their different structures will have phase differences caused both by variations in refractive index arising from changes in protoplasmic concentration and by differences in thickness.

    3. This is a specially designed light microscope in which annular phase plate and annular diaphragm (Fig. 148) are fitted.

    4. The annular diaphragm is situated below the condenser. It is made up of a circular disc having a circular annular groove. The light rays are allowed to pass through the annular groove. Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied.

    At the back focal plane of the objective develops an image. The annular phase plate is placed at this back focal plane. This phase plate is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular groove. This thick or thin area in the phase plate is called the conjugate area. The phase plate is a transparent disc.

    5. With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this microscope. This is obtained by separating the direct rays from the diffracted rays. The direct light rays pass through the annular groove whereas the diffracted light rays pass through the region outside the groove.

    6. Depending upon the different refractive Indices of the different cell components, the object to be studied shows different degree of contrast in this microscope.

    7. No special preparation of fixation or staining, etc. is needed for study an object under phase contrast microscope. This saves a lot of time of the researcher because a clear picture of unstained or living cells is easily seen under this microscope.

    8. Applications of phase contrast microscopy in biological research are numerous.

    By this, one can study:

    (i) The actual processes of mitosis and meiosis in living cells

    (ii) The actual effects of several chemicals on the living cells

    (iii) The behaviour of several microscopic organisms (e.g. Protozoans) towards various chemical and physical factors and

    (iv) Processes related to permeability of plasma membrane, etc.

    Type # 6. Interference Microscope:

    1. This is an improved type of phase contrast microscope which displays the interference between light transmitted and that not transmitted through a specimen.

    2. The interference colours produced in this microscope indicate differences in refractive index.

    3. The light in this microscope is split into two beams, of which one passes through the specimen and the other through a reference beam. The two beams are then made to interfere at the image plane. Areas of the specimen having similar optical paths appear in the image similarly coloured or similarly bright.

    4. Differing from the phase contrast microscope, in interference microscope the central wave and the diffracted wave are completely separated while passing through the phase plate.

    5. In this microscope, the annular phase plate is fixed below the sub-stage condenser while the phase plate is fixed above the objective lens in the objective. The ocular lens is used for observing the image.

    6. This microscope can be used to measure the thickness of the cell and its components. It is also used to determine the dry weight of microscopic objects such as proteins, nucleic acid, etc.

    Type # 7. Differential Interference Contrast Micro­scope:

    1. Also called DIC microscope or Nomarski microscope, this is an improved version of interference microscope.

    2. In the optical system of this microscope only a single light beam is used.

    3. Passing through the object, objective and a special birefringent prism, the single light beam gets divided into interfering beams.

    4. A polarizer, analyzer filters and a compensating prism are also present in this microscope.

    5. Excellent contrast of edges of cells and their organelles are seen under this microscope, and their visual effect is three-dimensional.

    6. Similar to phase contrast microscope, only unstained cells or materials are studied by this microscope.

    Type # 8. Fluorescent Microscope:

    1. This is an advance type of light microscope in which the specimen is irradiated at wavelengths which will excite the natural or artificially introduced fluorochromes. An optical filter of this microscope absorbs the exciting wavelengths but transmits the fluorescent image which can be studied normally.

    2. It is a microscope used for observing fluorescence in cells and tissues. (Fluorescence is a pheno­menon in which the wavelength of invisible ultraviolet light is converted into a wavelength of light in the visible range).

    3. A fluorescent microscope consists of (i) a lamp, (ii) the optical system, and (iii) a system of observation.

    4. Ultraviolet (UV) light is used as a source of illumination in this microscope. For getting UV rays, high pressure mercury arcs lamps, xenon arcs lamps or tungsten halogen lamps are used. A pair of adaptors (complementary filters) are also fitted in it. Since UV rays are harmful to human eye, the ocular lens used in this microscope is made of ordinary glass. This helps in preventing UV rays reaching the eye, but the fluorescence can be observed easily.

    5. This microscope can detect fluorescent materials (e.g. dyes) easily in the cells, even if they are present in traces.

    6. It is also used in the study of DNA, RNA, proteins and chromosome banding, localization of Y-chromosome in man, detection of heterochromatin and in several studies of immunology.

    Type # 9. Electron Microscope:

    1. The electron microscope is an instrument which utilizes the short wavelength of an electron beam, rather than light waves, to obtain very high magnification and resolution of minute structures for which a light microscope is inadequate. It contains an electric gun whose beam is refracted and focused onto a specimen by an electron lens system. The image of the specimen is magnified and projected onto a stage or fluorescent screen.

    2. On the basis of several earlier researches in Physics, Knoll and Ruska (1931) of Berlin were the first to develop an electron microscope. With the help of their electron microscope, they could magnify objects up to 12,000 times.

    By an Improved type of electron microscope developed by Borries and Ruska (1938), they could obtain pictures of 20,000 times magnification. In this die magnification of objective coil was 100 and the projector coil was 200. The resolution of this microscope was 100Å.

    The electron microscopes with 4-1 OÅ or even better resolution are now available.

    3. A magnification as high as 1, 60,000 times can be achieved by vising intermediate coils between the objective and projector coils of the electron microscope.

    4. Some of the basic differences (Fig. 149) between a light microscope and an electron microscope are mentioned in Table 13.1.

    Some differences between light micro­scope and electron microscope:

    1. Visible light is used in this microscope.

    2. Source of illumi­nation is situated at the bottom.

    3- For magnification in this microscope the lens system consists of glass lenses,

    4. The lenses arc ocular, objective and conden­ser.

    5. The image is either seen with the eye or recorded on a photo­graphic film with a camera in this microscope.

    1. Electrons are used in electron microscope.

    2. Source of illumination is situated at the top in this microscope.

    3. The lens system consists of electromagnetic coils in this microscope.

    4. This microscope has projector coils, an objec­tive and a condenser.

    5. The image in an electron microscope is either recorded on a fluore­scent screen or recorded on a photographic film.

    An electron microscope consists of an electric gun, microscope column, electromagnetic coils, a fluorescent screen and some other accessories described below:

    (a) The electron gun is located at the top of the body of microscope. It is the source of electrons. It is made up of a tungsten filament surrounded by a negatively biased shield with an aperture. The electron beam is drawn off through this aperture.

    (b) The microscope column or central column is made up of an evacuated metal tube. It protects the person operating the microscope from X-rays that are generated when the electrons strike the surface of the metal tube.

    (c) The electromagnetic coils or lenses include projector coils, objective and condenser. In each coil, the coils of electric wire are wound on a hollow metallic cylinder. The magnetic field, produced by passing the electric current through the magnetic coil, functions as a magnifying lens.

    (d) The fluorescent screen is used for observing the magnified image of the object. It remains coated with a chemical which, on being excited, forms the image as on the screen of television.

    (e) Some other essential accessories of the electron microscope Include high voltage transformers (for developing high voltage current for the electron gun and electromagnetic coils), vacuum pumps (for maintaining high vacuum inside the microscope column), a water cooling system (for prevention from overheating of various parts), a circulating pump, a refrigeration plant and also’ a filter system.

    All these parts require elaborate arrangements and contribute to the massive size of the electron microscope.

    6. The image formation in this microscope occurs by the scattering of electrons. The electrons strike the atomic nuclei and get dispersed. These dispersed electrons form the electron image. By projecting on a fluorescent screen or photographic film, this electron image is converted into a visible image of the object.

    7. The electron beam in this microscope is made by accelerating electrons through a potential difference of from 1-1500kV in an electron gun.

    8. Only dried specimens are studied by electron microscope. Living cells cannot be studied with this microscope because they possess water which causes large scale scattering of electrons.

    9. Ultra thin sections (10-50 nm thickness), which are more than 200 times thinner than those routinely used for light microscopy, are cut for electron microscopy. These are cut with the help of diamond or glass knives of an ultra-microtome.

    Type # 10. Transmission Electron Microscope or TEM:

    1. Transmission electron microscope or TEM (Fig. 8) is a form of electron microscope in which the electrons are allowed to be transmitted through the objects.

    2. The specimen to be studied is evenly illuminated by a broad beam of electrons at 40-100kV, and the image is formed directly by focusing those electrons which pass more or less un-scattered through the specimen either on a fluorescent screen or on a photographic film.

    3. This microscope has a fine resolution of 0.3 nm or less but is usually not suitable for living specimens.

    Type # 11. Scanning Electron Microscope or SEM:

    1. This is also a form of electron microscope in which an extremely fine beam of electrons is made at 3-30kV for scanning a selected or specific area of specimen. This is actually a combination of the technology of electron microscopy and television electronics.

    2. The specimen to be examined is usually a solid object. The secondary electrons, which are emitted from or near the surface, are collected and analyzed. After analysis, they form a signal which modulates the beam of a cathode ray tube (Fig. 151) scanned in synchrony with a secondary beam.

    3. The images, formed in this microscope, resemble those seen in a hand lens. They can be magnified about 100,000 times.

    4. This microscope is extremely useful for studying surface structures of thick specimens, cells, tissues and membranes.

    5. Because the electrons in this microscope form the image by getting emitted from the surface of the specimen, the image provides a three- dimensional appearance. This three-dimensional image is formed by secondary electrons which are first collected, amplified and then used for the image formation on the phosphor screen of a cathode ray tube.

    6. At least two cathode ray tubes are present in this microscope, of which one is used for visual observation of the specimen and the other is used for photography.

    Type # 12. Scanning Transmission Electron Micro­scope or STEM:

    1. In scanning transmission electron microscope or STEM, the specimen is scanned in the same way as in scanning electron microscope or SEM but transmitted electrons are collected and utilized for the formation of picture on the screen.

    2. The main advantage of this electron microscope is that it is fitted with detector systems. Due to this facility, it can separately detect the scattered electrons, un-scattered electrons, and also those electrons which are there as a result of the combination of scattered and un-scattered electrons.

    Type # 13. Polarization Microscope:

    1. This is a special kind of light microscope fitted with a polarizer and an analyzer.

    2. Plane-polarized light is utilized in this microscope for studying the presence of preferentially-oriented constituents, their shape, the direction of their orientation and their refractive index in the cells and tissues.

    3. The principle of this microscope is based on the behaviour of some components of cell when observed under polarized light.

    4. The polarizer and analyzer, the two main components of this microscope, are either made of nicol prism or a polaroid disc. Above the polarizer is fitted the condenser. The analyzer is located above the objective. A compensator or gypsum plate is also inserted in between the polarizer and analyzer.

    5. This microscope is also used for both quantitative as well as qualitative studies in several biological disciplines including cytology, anatomy, histology and pathology.


    4.3 Eukaryotic Cells

    By the end of this section, you will be able to do the following:

    • Describe the structure of eukaryotic cells
    • Compare animal cells with plant cells
    • State the role of the plasma membrane
    • Summarize the functions of the major cell organelles

    Have you ever heard the phrase “form follows function?” It’s a philosophy that many industries follow. In architecture, this means that buildings should be constructed to support the activities that will be carried out inside them. For example, a skyscraper should include several elevator banks. A hospital should have its emergency room easily accessible.

    Our natural world also utilizes the principle of form following function, especially in cell biology, and this will become clear as we explore eukaryotic cells (Figure 4.8). Unlike prokaryotic cells, eukaryotic cells have: 1) a membrane-bound nucleus 2) numerous membrane-bound organelles such as the endoplasmic reticulum, Golgi apparatus, chloroplasts, mitochondria, and others and 3) several, rod-shaped chromosomes. Because a membrane surrounds eukaryotic cell’s nucleus, it has a “true nucleus.” The word “organelle” means “little organ,” and, as we already mentioned, organelles have specialized cellular functions, just as your body's organs have specialized functions.

    At this point, it should be clear to you that eukaryotic cells have a more complex structure than prokaryotic cells. Organelles allow different functions to be compartmentalized in different areas of the cell. Before turning to organelles, let’s first examine two important components of the cell: the plasma membrane and the cytoplasm.

    Visual Connection

    If the nucleolus were not able to carry out its function, what other cellular organelles would be affected?

    The Plasma Membrane

    Like prokaryotes, eukaryotic cells have a plasma membrane (Figure 4.9), a phospholipid bilayer with embedded proteins that separates the internal contents of the cell from its surrounding environment. A phospholipid is a lipid molecule with two fatty acid chains and a phosphate-containing group. The plasma membrane controls the passage of organic molecules, ions, water, and oxygen into and out of the cell. Wastes (such as carbon dioxide and ammonia) also leave the cell by passing through the plasma membrane.

    The plasma membranes of cells that specialize in absorption fold into fingerlike projections that we call microvilli (singular = microvillus) (Figure 4.10). Such cells typically line the small intestine, the organ that absorbs nutrients from digested food. This is an excellent example of form following function. People with celiac disease have an immune response to gluten, which is a protein in wheat, barley, and rye. The immune response damages microvilli, and thus, afflicted individuals cannot absorb nutrients. This leads to malnutrition, cramping, and diarrhea. Patients suffering from celiac disease must follow a gluten-free diet.

    The Cytoplasm

    The cytoplasm is the cell's entire region between the plasma membrane and the nuclear envelope (a structure we will discuss shortly). It is comprised of organelles suspended in the gel-like cytosol , the cytoskeleton, and various chemicals (Figure 4.8). Even though the cytoplasm consists of 70 to 80 percent water, it has a semi-solid consistency, which comes from the proteins within it. However, proteins are not the only organic molecules in the cytoplasm. Glucose and other simple sugars, polysaccharides, amino acids, nucleic acids, fatty acids, and derivatives of glycerol are also there. Ions of sodium, potassium, calcium, and many other elements also dissolve in the cytoplasm. Many metabolic reactions, including protein synthesis, take place in the cytoplasm.

    The Nucleus

    Typically, the nucleus is the most prominent organelle in a cell (Figure 4.8). The nucleus (plural = nuclei) houses the cell’s DNA and directs the synthesis of ribosomes and proteins. Let’s look at it in more detail (Figure 4.11).

    The Nuclear Envelope

    The nuclear envelope is a double-membrane structure that constitutes the nucleus' outermost portion (Figure 4.11). Both the nuclear envelope's inner and outer membranes are phospholipid bilayers.

    The nuclear envelope is punctuated with pores that control the passage of ions, molecules, and RNA between the nucleoplasm and cytoplasm. The nucleoplasm is the semi-solid fluid inside the nucleus, where we find the chromatin and the nucleolus.

    Chromatin and Chromosomes

    To understand chromatin, it is helpful to first explore chromosomes , structures within the nucleus that are made up of DNA, the hereditary material. You may remember that in prokaryotes, DNA is organized into a single circular chromosome. In eukaryotes, chromosomes are linear structures. Every eukaryotic species has a specific number of chromosomes in the nucleus of each cell. For example, in humans, the chromosome number is 46, while in fruit flies, it is eight. Chromosomes are only visible and distinguishable from one another when the cell is getting ready to divide. When the cell is in the growth and maintenance phases of its life cycle, proteins attach to chromosomes, and they resemble an unwound, jumbled bunch of threads. We call these unwound protein-chromosome complexes chromatin (Figure 4.12). Chromatin describes the material that makes up the chromosomes both when condensed and decondensed.

    The Nucleolus

    We already know that the nucleus directs the synthesis of ribosomes, but how does it do this? Some chromosomes have sections of DNA that encode ribosomal RNA. A darkly staining area within the nucleus called the nucleolus (plural = nucleoli) aggregates the ribosomal RNA with associated proteins to assemble the ribosomal subunits that are then transported out through the pores in the nuclear envelope to the cytoplasm.

    Ribosomes

    Ribosomes are the cellular structures responsible for protein synthesis. When we view them through an electron microscope, ribosomes appear either as clusters (polyribosomes) or single, tiny dots that float freely in the cytoplasm. They may be attached to the plasma membrane's cytoplasmic side or the endoplasmic reticulum's cytoplasmic side and the nuclear envelope's outer membrane (Figure 4.8). Electron microscopy shows us that ribosomes, which are large protein and RNA complexes, consist of two subunits, large and small (Figure 4.13). Ribosomes receive their “orders” for protein synthesis from the nucleus where the DNA transcribes into messenger RNA (mRNA). The mRNA travels to the ribosomes, which translate the code provided by the sequence of the nitrogenous bases in the mRNA into a specific order of amino acids in a protein. Amino acids are the building blocks of proteins.

    Because protein synthesis is an essential function of all cells (including enzymes, hormones, antibodies, pigments, structural components, and surface receptors), there are ribosomes in practically every cell. Ribosomes are particularly abundant in cells that synthesize large amounts of protein. For example, the pancreas is responsible for creating several digestive enzymes and the cells that produce these enzymes contain many ribosomes. Thus, we see another example of form following function.

    Mitochondria

    Scientists often call mitochondria (singular = mitochondrion) “powerhouses” or “energy factories” of both plant and animal cells because they are responsible for making adenosine triphosphate (ATP), the cell’s main energy-carrying molecule. ATP represents the cell's short-term stored energy. Cellular respiration is the process of making ATP using the chemical energy in glucose and other nutrients. In mitochondria, this process uses oxygen and produces carbon dioxide as a waste product. In fact, the carbon dioxide that you exhale with every breath comes from the cellular reactions that produce carbon dioxide as a byproduct.

    In keeping with our theme of form following function, it is important to point out that muscle cells have a very high concentration of mitochondria that produce ATP. Your muscle cells need considerable energy to keep your body moving. When your cells don’t get enough oxygen, they do not make much ATP. Instead, producing lactic acid accompanies the small amount of ATP they make in the absence of oxygen.

    Mitochondria are oval-shaped, double membrane organelles (Figure 4.14) that have their own ribosomes and DNA. Each membrane is a phospholipid bilayer embedded with proteins. The inner layer has folds called cristae. We call the area surrounded by the folds the mitochondrial matrix. The cristae and the matrix have different roles in cellular respiration.

    Peroxisomes

    Peroxisomes are small, round organelles enclosed by single membranes. They carry out oxidation reactions that break down fatty acids and amino acids. They also detoxify many poisons that may enter the body. (Many of these oxidation reactions release hydrogen peroxide, H2O2, which would be damaging to cells however, when these reactions are confined to peroxisomes, enzymes safely break down the H2O2 into oxygen and water.) For example, peroxisomes in liver cells detoxify alcohol. Glyoxysomes, which are specialized peroxisomes in plants, are responsible for converting stored fats into sugars. Plant cells contain many different types of peroxisomes that play a role in metabolism, pathogene defense, and stress response, to mention a few.

    Vesicles and Vacuoles

    Vesicles and vacuoles are membrane-bound sacs that function in storage and transport. Other than the fact that vacuoles are somewhat larger than vesicles, there is a very subtle distinction between them. Vesicle membranes can fuse with either the plasma membrane or other membrane systems within the cell. Additionally, some agents such as enzymes within plant vacuoles break down macromolecules. The vacuole's membrane does not fuse with the membranes of other cellular components.

    Animal Cells versus Plant Cells

    At this point, you know that each eukaryotic cell has a plasma membrane, cytoplasm, a nucleus, ribosomes, mitochondria, peroxisomes, and in some, vacuoles, but there are some striking differences between animal and plant cells. While both animal and plant cells have microtubule organizing centers (MTOCs), animal cells also have centrioles associated with the MTOC: a complex we call the centrosome. Animal cells each have a centrosome and lysosomes whereas, most plant cells do not. Plant cells have a cell wall, chloroplasts and other specialized plastids, and a large central vacuole whereas, animal cells do not.

    The Centrosome

    The centrosome is a microtubule-organizing center found near the nuclei of animal cells. It contains a pair of centrioles, two structures that lie perpendicular to each other (Figure 4.15). Each centriole is a cylinder of nine triplets of microtubules.

    The centrosome (the organelle where all microtubules originate) replicates itself before a cell divides, and the centrioles appear to have some role in pulling the duplicated chromosomes to opposite ends of the dividing cell. However, the centriole's exact function in cell division isn’t clear, because cells that have had the centrosome removed can still divide, and plant cells, which lack centrosomes, are capable of cell division.

    Lysosomes

    Animal cells have another set of organelles that most plant cells do not: lysosomes. The lysosomes are the cell’s “garbage disposal.” In plant cells, the digestive processes take place in vacuoles. Enzymes within the lysosomes aid in breaking down proteins, polysaccharides, lipids, nucleic acids, and even worn-out organelles. These enzymes are active at a much lower pH than the cytoplasm's. Therefore, the pH within lysosomes is more acidic than the cytoplasm's pH. Many reactions that take place in the cytoplasm could not occur at a low pH, so again, the advantage of compartmentalizing the eukaryotic cell into organelles is apparent.

    The Cell Wall

    If you examine Figure 4.8, the plant cell diagram, you will see a structure external to the plasma membrane. This is the cell wall , a rigid covering that protects the cell, provides structural support, and gives shape to the cell. Fungal and some protistan cells also have cell walls. While the prokaryotic cell walls' chief component is peptidoglycan, the major organic molecule in the plant (and some protists') cell wall is cellulose (Figure 4.16), a polysaccharide comprised of glucose units. Have you ever noticed that when you bite into a raw vegetable, like celery, it crunches? That’s because you are tearing the celery cells' rigid cell walls with your teeth.

    Chloroplasts

    Like the mitochondria, chloroplasts have their own DNA and ribosomes, but chloroplasts have an entirely different function. Chloroplasts are plant cell organelles that carry out photosynthesis. Photosynthesis is the series of reactions that use carbon dioxide, water, and light energy to make glucose and oxygen. This is a major difference between plants and animals. Plants (autotrophs) are able to make their own food, like sugars used in cellular respiration to provide ATP energy generated in the plant mitochondria. Animals (heterotrophs) must ingest their food.

    Like mitochondria, chloroplasts have outer and inner membranes, but within the space enclosed by a chloroplast’s inner membrane is a set of interconnected and stacked fluid-filled membrane sacs we call thylakoids (Figure 4.17). Each thylakoid stack is a granum (plural = grana). We call the fluid enclosed by the inner membrane that surrounds the grana the stroma.

    The chloroplasts contain a green pigment, chlorophyll , which captures the light energy that drives the reactions of photosynthesis. Like plant cells, photosynthetic protists also have chloroplasts. Some bacteria perform photosynthesis, but their chlorophyll is not relegated to an organelle.

    Evolution Connection

    Endosymbiosis

    We have mentioned that both mitochondria and chloroplasts contain DNA and ribosomes. Have you wondered why? Strong evidence points to endosymbiosis as the explanation.

    Symbiosis is a relationship in which organisms from two separate species depend on each other for their survival. Endosymbiosis (endo- = “within”) is a mutually beneficial relationship in which one organism lives inside the other. Endosymbiotic relationships abound in nature. We have already mentioned that microbes that produce vitamin K live inside the human gut. This relationship is beneficial for us because we are unable to synthesize vitamin K. It is also beneficial for the microbes because they are protected from other organisms and from drying out, and they receive abundant food from the environment of the large intestine.

    Scientists have long noticed that bacteria, mitochondria, and chloroplasts are similar in size. We also know that bacteria have DNA and ribosomes, just like mitochondria and chloroplasts. Scientists believe that host cells and bacteria formed an endosymbiotic relationship when the host cells ingested both aerobic and autotrophic bacteria (cyanobacteria) but did not destroy them. Through many millions of years of evolution, these ingested bacteria became more specialized in their functions, with the aerobic bacteria becoming mitochondria and the autotrophic bacteria becoming chloroplasts.

    The Central Vacuole

    Previously, we mentioned vacuoles as essential components of plant cells. If you look at Figure 4.8b, you will see that plant cells each have a large central vacuole that occupies most of the cell's area. The central vacuole plays a key role in regulating the cell’s concentration of water in changing environmental conditions. Have you ever noticed that if you forget to water a plant for a few days, it wilts? That’s because as the water concentration in the soil becomes lower than the water concentration in the plant, water moves out of the central vacuoles and cytoplasm. As the central vacuole shrinks, it leaves the cell wall unsupported. This loss of support to the plant's cell walls results in the wilted appearance.

    The central vacuole also supports the cell's expansion. When the central vacuole holds more water, the cell becomes larger without having to invest considerable energy in synthesizing new cytoplasm.


    Godfrey Hounsfield and Allan Cormack develop the computerised axial tomography (CAT) scanner. With the help of a computer, the device combines many X-ray images to generate cross-sectional views as well as three-dimensional images of internal organs and structures.

    John Venables and CJ Harland observe electron backscatter patterns (EBSP) in the scanning electron microscope. EBSP provide quantitative microstructural information about the crystallographic nature of metals, minerals, semiconductors and ceramics.


    Talk Overview

    This talk introduces two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.

    Questions

    1. Explain why only red light is emitted through thin skin when a flashlight is shone on it.
    2. What is clearing and what is it used for?
    3. What are the advantages of using two infrared photons instead of one blue photon in two-photon microscopy to excite a green-light emitting molecule?
    4. Why does two-photon microscopy not require a pinhole (select all that apply)?
      1. Because the emitted light in two-photon microscopy does not scatter
      2. Because there is no out-focus light in two-photon microscopy
      3. Because two-photon microscopy gives rise to localized excitation
      4. None of the above
      1. The detected image would include less background signal
      2. The detected image would be dimmer
      3. None of the above

      Answers

      1. Because biological materials absorb light in most wavelengths, except in the red color. Therefore, more red light is let through the skin than the other wavelengths
      2. Clearing removes scattering from biological samples. The solution uses vary and include improving “deep” imaging in thick fixed samples. The clearing solutions are either organic solvents, detergents, or urea.
      3. Using two photons instead of one will increase penetration depth Because biological molecules do not usually absorb light in the red spectrum, using red photons to excite fluorescent markers can decrease the image background In addition, two red photons used simultaneously can excite a fluorescent marker that is usually excited in the blue wavelength (in one-photon microscopy). This increases the range of fluorescent markers that can be used in thick sections Last, because two-photon microscopy involves localized excitation, it does not involve pinholes.
      4. B and C
        1. Incorrect: The emitted light in two-photon microscopy does scatter, but it does not matter. Because only one point emits light (the focus of the light beam), the total emission is measured.
        2. Correct: Because there is no out-focus light in two-photon microscopy, the pinhole is not required in two-photon microscopy.
        3. Correct: Two-photon microscopy gives rise to localized excitation, which means that only the focus point receives sufficient light (from two photons) to be excited and emit light. Therefore, the excitation photons cannot produce sufficient light to excite markers, which means that there is no background light to filter through a pinhole.
        1. Incorrect: The detected image would include less background signal. There is no background signal in two-photon microscopy
        2. Correct: The detected image would be dimmer. Because the total emitted light is measured in two-photon microscopy, and because the pinhole would filter out a significant portion of that emitted light, light would be lost if the pinhole were to be kept on.)

        DIC Microscope Configuration and Alignment

        Differential interference contrast (DIC) optical components can be installed on virtually any brightfield transmitted, reflected, or inverted microscope, provided the instrument is able to accept polarizing filters and the specially designed condenser and objective prisms (together with the housings) necessary to perform the technique.

        All of the major microscope manufacturers produce DIC accessories for their research-level microscopes, and these are often bundled together as matched kits containing all of the required hardware and optical components. In the standard configuration, a differential interference contrast microscope (see Figure 1) contains the polarizing elements typically encountered on a polarized light microscope and, in addition, two specially constructed birefringent compound prisms. Termed Wollaston or Nomarski prisms, these optical beamsplitters (and beam combiners) are positioned to project interference patterns of sheared wavefronts into the condenser front focal plane and the objective rear focal plane.

        Presented in Figure 1 is the typical optical train configuration for a transmitted differential interference contrast microscope. Semi-coherent wavefronts emitted from localized neighborhoods in the lamp filament pass through a linear polarizer positioned between the light port in the microscope base and the condenser assembly. A compound Nomarski or Wollaston prism is located in or near the condenser aperture (front focal plane), and serves to align and shear incident polarized wavefronts into two orthogonal components. The perpendicular, sheared wavefronts are focused by the condenser lens system into parallel bundles that traverse the specimen plane and respond to refractive index and thickness gradients by deformation according to optical path length parameters of the specimen.

        Light waves gathered by the objective converge at the rear focal plane where a second Nomarski prism is located. The objective Nomarski prism recombines sheared and deformed wavefronts into linear and elliptically polarized light that subsequently passes a component through the analyzer (a second polarizer oriented perpendicular to the substage polarizer) located in an intermediate tube above the objective. Finally, the linear polarized light components that emerge from the analyzer recombine through constructive and destructive interference at the image plane in the eyepiece diaphragm (or camera projection lens).

        Polarizing Elements for DIC Microscopy

        The polarizers employed in DIC microscopy are similar or identical to those used for polarized light observations, but many manufacturers offer matched DIC polarizing filters that have both low and high light transmission efficiencies. At the upper end of the magnification range (40x, 60x, and 100x), polarizers with high transmission efficiencies are preferred for DIC because specimen details are more clearly delineated and larger bias retardation values can be utilized for video and digital imaging. The low transmission polarizing filters are useful for DIC observation using the 10x and 20x objectives, but severely restrict light transmission at higher magnifications.

        On most microscopes, the polarizer is mounted directly onto the light port in the base of the instrument or in a filter holder attached underneath the condenser. Polarizers designed to span the light port generally are mounted in a rotating assembly that enables the microscopist to rotate the filter through a 90 to 180-degree angle (see Figure 2(a)). Once the rotating polarizer is positioned at the desired transmission azimuth, it can be secured into place with a locking screw. By convention, the polarizer is oriented with the vibration transmission axis positioned in an East-West direction (left to right when standing in front of the microscope). Microscopes that have the polarizer attached to the substage condenser mounting bracket are provided with a housing that may or may not enable rotation of the polarizer (Figures 2(b) and 2(c)). Polarizers having circular geometry fit into a bracket in the stage and can rotate in fixed increments of 45 degrees with a détente. Other polarizers are mounted in rectangular frames that slide into a slot in the condenser bracket (Figures 2(b) and 2(c)), and often contain thumbwheels that enable rotation through 180 to 360 degrees.

        Analyzers are placed between the objective Nomarski prism and the eyepiece observation tubes in microscopes equipped for DIC observation, similar to the location of these components in polarized light microscopes. The analyzer is also a linear polarizer that is positioned with the transmission azimuth oriented at a 90-degree angle with respect to that of the polarizer. By convention, the vibration direction for the analyzer is North-South, which coincides with the East-West orientation prescribed for the polarizer.

        The microscope mounting configuration for analyzers is equally as varied as it is for polarizers, and these components are commonly inserted into the optical train throughout a number of locations. In some microscopes, the analyzer is fixed into a rectangular frame (Figure 2(e)) and inserted into a slot in the nosepiece, intermediate tube, or vertical illuminator. Other analyzer designs feature the same frame style, but enable rotation of the analyzer element with a thumbwheel that is often graduated in 10, 45, or 90-degree increments (Figure 2(f)). Polarized light microscopes equipped for DIC observation often house the analyzer in an intermediate tube (see Figure 2(d)) located between the objective nosepiece and the observation tubes. These units are often designed for precision measurements in polarized light and feature 360-degree graduated vernier scales wrapped around the circumference, with a locking mechanism to secure the analyzer in the desired transmission azimuth. In addition, the analyzer is usually mounted on a slider so that it can be conveniently removed from the light path for linearly polarized or brightfield observation. Intermediate tubes for polarized light microscopy also contain a 20 × 6 millimeter DIN standard slot for a quarter-wavelength, full-wave, or de Sénarmont compensator (Figure 2(d) and Figure 5).

        Modern microscopes position the polarizer and analyzer in strategic locations with respect to the field lens, condenser, objective and observation tubes. In older microscopes, these polarizing elements can be found installed in a wide variety of locations. It should be noted, however, that placing polarizing elements in or very near a conjugate image plane (the field diaphragm, specimen stage, or eyepiece fixed aperture) is not a good idea, because scratches, imperfections, dirt, and debris on the glass surface can be imaged along with the specimen.

        Condenser and Objective Prisms

        DIC condenser prisms, which act as beamsplitters to produce an angular shear to incoming polarized light wavefronts, are often mounted in a revolving turret condenser designed to house at least three individual prisms, as illustrated in Figure 3. Turret specifications and configurations vary according to the manufacturer, but they generally contain slots for four to eight auxiliary components, including Wollaston or Nomarski prisms, phase contrast annular rings, or darkfield light stops. The condenser turret illustrated in Figure 3 contains seven openings, three of which are filled with phase contrast annuli and three with DIC prisms. The open slot is used for brightfield observation.

        Each condenser DIC prism (also termed compensators or auxiliary prisms) must be specifically matched to a narrow range of objective numerical apertures, so a particular prism may only work for one or two objectives (for example, the 10x and 20x). As a result, between three and five condenser prisms must be employed to match the entire objective magnification range between 10x and 100x. Some manufacturers tailor each condenser prism for a particular objective, thus requiring up to seven condenser prisms to span the entire spectrum of dry and oil immersion objectives having varied numerical apertures.

        Condenser DIC prism inserts are constructed with anodized circular aluminum plates having the combination prism (usually circular in shape) secured in a precise orientation with optical cement. DIC prism wedges are very thin and cut with close tolerances to ensure that angular shear values match those required by the objective numerical aperture. The polished plates must be handled carefully to avoid contamination from fingerprints, oil, dirt, and debris. Each prism frame contains a slot or pin that mates to a corresponding partner in the condenser turret in order to define and secure alignment of the condenser prism with respect to the objective prism and the polarizer (and analyzer) axes.

        Positioned between the objective and the analyzer is a second Nomarski compound prism that acts to recombine wavefronts sheared by the condenser prism (Figures 1, 4, and 5). This prism, often termed the objective or principal DIC prism, can either be matched to a specific condenser prism, or a single objective prism can serve to recombine wavefronts having the spectrum of shear angles represented by all of the condenser prisms. A majority of the microscope manufacturers configure their DIC microscopes to employ a single objective Nomarski prism, which is mounted in a rectangular frame that slides into the nosepiece (see Figure 4). In order to introduce bias retardation into the DIC optical system, the objective prism rides on a gliding support that can be translated back and forth across the microscope optical axis by means of a micrometer control knob.

        Objective DIC prisms designed to introduce bias retardation through translation across the microscope optical axis are fabricated in a rectangular shape with the long axis corresponding to direction of prism shear (Figure 4(b)). In a properly aligned DIC microscope, the condenser prism is imaged onto the objective prism by the combined action of the condenser and objective lens systems. As a result, the wavefront shear produced by the condenser prism is exactly matched at every point along the surface of both prisms (which are inverted with respect to one another). Translating either prism along the shear axis produces a wavefront mismatch (bias retardation), which, in turn, introduces an optical path difference that is uniform across the microscope aperture. Translating the objective prism with the micrometer control is the most popular mechanism for producing bias retardation in DIC microscopes.

        Several objective prism slider designs from various manufacturers are illustrated in Figure 4. All of the compound prisms are housed in rectangular frames with a translational control knob positioned at the end of the frame. The control knob is employed to shift the prism position laterally along the shear axis (Figure 4(b)) in order to introduce bias retardation (or a net wavefront optical path difference), into the differential interference contrast optical train. Nomarski prism sliders designed for reflected light DIC microscopy also contain a second control knob (Figure 4(c) and 4(d)) that enables adjustment of the prism height to match varying rear focal plane positions throughout the entire objective magnification and numerical aperture range. When the microscope is operated in another imaging mode, the objective prism frame can be conveniently removed from the optical path by sliding the entire frame without removing it from the nosepiece or intermediate tube housing.

        In DIC microscopes designed to introduce bias retardation using a de Sénarmont compensator, the objective Nomarski prisms are secured with fixed mounts that slide into the nosepiece above the objectives (Figure 5(c)). Microscopes using de Sénarmont compensators require a separate objective prism for each objective, but can often utilize the same condenser Nomarski prism for two or more objectives. The fixed objective prism mounts (Figure 5(c)) are easily removed from the light path by sliding the frame away from the microscope nosepiece.

        A typical polarizer and quarter-wavelength retardation plate configuration for de Sénarmont differential interference contrast is illustrated in Figure 5(a). This unit is fitted over the field diaphragm adjustment knob on the microscope base and, after alignment of the retardation plate optical axis and polarizer transmission azimuth, secured to the knob with a locking setscrew. The polarizer axis is marked on the front of the de Sénarmont compensator unit, and graduated rulings enable the operator to qualitatively determine the approximate amount of bias retardation introduced into the system when the polarizer is rotated. The locking knob can be utilized to hold the polarizer immobile with respect to the quarter-wavelength plate. For brightfield observation or enhanced contrast techniques without DIC, the entire polarizer and retardation plate assembly can be removed from the optical path by swinging out the hinged upper section.

        An alternative technique for de Sénarmont DIC compensation is to place the quarter-wavelength compensator plate in the optical train between the objective Nomarski prism and the analyzer. In this case, either a standard linear polarizer is employed beneath the condenser prisms or the de Sénarmont compensator illustrated in Figure 5(a) is set with the polarizer axis parallel to the retardation plate fast axis (only linear polarized light emerges from the compensator) and perpendicular to the analyzer. An intermediate tube designed for introducing de Sénarmont DIC compensation after the objective prism is illustrated in Figure 5(b). In this case, bias retardation is introduced into the optical system by rotating the analyzer, rather than the polarizer.

        Equipped with a Bertrand lens for observing the objective rear focal plane, the intermediate tube presented in Figure 5(b) also has a DIN standard slot able to accommodate a variety of retardation plates, including a de Sénarmont compensator (shown in the Figure). The analyzer can be rotated by a precision graduated vernier mechanism, which enables the operator to quantitatively determine the level of bias retardation introduced into the DIC optical system. With de Sénarmont compensators, bias retardation values ranging between one-twentieth and a full wavelength can be easily measured with an accuracy of 0.15 nanometers. In addition, the Bertrand lens can be conveniently inserted into the light pathway by means of a thumbwheel in order to observe events occurring at the objective rear focal plane. The use of a Bertrand lens or phase telescope to monitor the objective focal plane is critical to DIC microscope alignment, as discussed below.

        In DIC microscopy, contrast is a function of specimen orientation, and the wide variety of geometries encountered often requires repositioning of the specimen to maximize contrast effects for observation of the target structures. Use of standard rectangular mechanical stages to observe DIC specimen is hampered by the limited range of rotational motion exhibited by these components, although the x-y translation mechanism eases the burden of carefully examining the entire contents of a microscope slide. In order to allow specimens to be easily rotated and manipulated to present the optimum azimuthal contrast, a circular rotation stage similar to that illustrated in Figure 6(a) is recommended for DIC microscopy. The circular stage enables 360-degree rotation of the specimen, which is often necessary with extended linear specimens (such as diatoms or filamentous structures) that display extreme azimuthal contrast effects in DIC. Translation of the specimen during observation is aided by the utilization of a graduated mechanical stage accessory (Figure 6(b)) that attaches directly to the circular stage and securely clamps a microscope slide. The stage should be centered along the microscope optical axis, either by adjusting the position of the stage itself or by centering the objectives and condenser with respect to the stage.

        Objectives designed for differential interference contrast must be free of strain or birefringent occlusions that depolarize light and lead to image degradation. In the past, microscopists were limited to strain-free achromat and fluorite objectives intended for observations in polarized light, but modern objectives having higher correction factors can now be employed for DIC microscopy. Termed universal objectives, these advanced lens systems can often be used for combined DIC, fluorescence, phase contrast, and brightfield microscopy without having to change objectives. In addition, newer apochromatic objectives have been designed that are sufficiently strain free to enable their use in polarized light and differential interference contrast observation, dramatically improving the image quality and resolution at higher magnifications.

        Differential Interference Contrast Microscope Alignment

        Before attempting to configure a microscope for observation in differential interference contrast, inspect the instrument to ascertain whether all of the necessary components are present, and free of lint, dust, and debris. Objective and condenser lens elements that contain stress signatures can degrade images in DIC, as can dirty lens surfaces, scratches, and contaminating foreign material in the optical pathway. Proper alignment of the microscope is essential to achieving optimum results and producing images that display pseudo three-dimensional and shadow-cast effects. Many of the steps outlined in the following procedure are only necessary when first aligning the microscope for DIC and do not require repetition for daily observations. Other steps should be taken each time the microscope is used for DIC investigations.

        Preliminary Microscope Inspection - Examine the microscope carefully to ensure that all necessary DIC components are installed, or available and ready for use when necessary. Remove the condenser, disassemble the turret, and inspect the condition of the Nomarski or Wollaston prisms. The surfaces of these compound prisms should be clean and free of dust and debris. Because they are housed within the condenser turret, DIC condenser prisms rarely become contaminated with fingerprints, but dust and lint can easily flow into the turret and land on one of the flat quartz surfaces. To clean a contaminated prism surface, use a rubber balloon to remove loose fibers and dust, and/or gently wipe the surface with lens tissue or moist soft cotton. Be careful not to scratch the surfaces. The same treatment should be afforded to the objective prism(s), condenser and objective external lens elements, microscope eyepiece lenses, and the field lens at the field diaphragm port in the base of the microscope (or attached to the pillar of an inverted microscope). After ensuring the critical components are clean, reassemble the microscope, install the polarizer and analyzer, and then align the optical system for Köhler illumination.

        Install the Polarizer and Analyzer - With the microscope disassembled (condenser, DIC prisms, and at least one objective removed), install the polarizer and analyzer in their positions beneath the condenser and above the objective, respectively. In a manner similar to polarized light microscopy, the polarizer and analyzer are positioned so their transmission azimuths are crossed at a 90-degree angle (perpendicular) to one another. The polarizer, which is mounted between the light source and the condenser, is traditionally oriented in an East-West direction, or left to right when facing the microscope. In some cases, the positions of both the polarizer and analyzer are pre-determined by their fixed position in the mounting frames, and these components can only be inserted into the microscope optical pathway with a single orientation. Usually, a marker on the polarizer mount indicates the transmission direction, but some microscopes are equipped with a rotating polarizer mount that is graduated in degrees (Figure 2). The analyzer may also be rotatable with a graduated knob, and/or may contain a mark indicating the transmission axis.

        When the polarizer and analyzer are crossed (transmission axes oriented at a 90-degree angle), the viewfield appears very dark when observed through the eyepieces. This condition is referred to as maximum extinction. If a significant amount of light passes through the microscope and the viewfield is not dark (or almost black), check to ensure the polarizer and analyzer are crossed. After crossing the polarizers, insert the condenser and objective, but do not install the objective Nomarski prism slider (or fixed mount). Rotate the condenser turret to the brightfield position (the slot lacking a phase plate or DIC prism). The viewfield should remain dark, but if either of these components (the condenser or objective) contains strained lens elements, some light may pass through. Remove one of the polarizing elements from the optical train (either the polarizer or analyzer) before proceeding to the next step.

        Establish Köhler Illumination - Before proceeding with DIC configuration (after the polarizers are installed), the microscope optical system should be aligned for brightfield specimen observation using the standard Köhler technique. When properly configured, an image of the light source (usually a tungsten-halogen lamp) should be projected onto the condenser aperture diaphragm plane by the collector lens housed in the lamphouse or along the optical train inside the microscope frame base. Simultaneously, the condenser lens system also projects an image of the field diaphragm into the specimen conjugate plane (at the microscope stage). After the lamp filament has been centered (most modern lamphouses contain a pre-centered lamp), close the condenser aperture diaphragm and align the condenser with the microscope optical axis in brightfield illumination (the condenser turret is set to the 0 or B position). Bring the diaphragm into focus, superimposed on a focused specimen using the 10x objective, and open the iris leaves until only a small portion of the diaphragm is visible at the peripheral edges of the viewfield. Similar steps are taken for each objective being used, making sure the microscope is properly configured for Köhler illumination for each objective in turn by adjusting both the field and aperture diaphragms. During the course of daily observations in DIC, the microscope should be periodically checked to ensure that Köhler illumination is maintained.

        Inspect the Objective Rear Aperture - After configuring the microscope for Köhler illumination, insert the polarizer and analyzer and examine the objective rear focal plane with a phase telescope or Bertrand lens (conoscopic observation mode). If the polarizer and analyzer are properly positioned and the microscope perfectly aligned, a dark extinction cross will appear in the objective aperture, as illustrated in Figure 7(a). The arms of the extinction cross should be oriented vertically and horizontally, with a small amount of light appearing at the four corners of the aperture (Figure 7(a)). Bright spots in the cross or highly birefringent regions, which affect the integrity of the extinction cross, are an indicator of strained optics. In addition, dust and lint particles positioned near a conjugate aperture focal plane (condenser or objective) will appear bright when viewed at the objective rear aperture. If birefringent spots are present, check another strain-free objective to determine whether the first objective or the condenser lens system is strained. Remove any contaminating dust from the objective or condenser lens surfaces and replace strained optical components (if possible) before proceeding to the next step.

        Objective DIC Prism Alignment - Install the objective prism by either inserting the slider (Figure 4) or a prism confined to a fixed mount (for systems utilizing de Sénarmont bias retardation see Figure 5). Once the prism is in position, examine the objective rear focal plane once again with the phase telescope or Bertrand lens. The viewfield should now appear very bright, but featureless, with a single dark interference fringe extending across the diameter of the aperture at a 45-degree angle (see Figure 7(b)) along the shear axis. Depending upon whether the microscope is upright or inverted, the interference fringe will traverse the objective rear aperture in a northeast-southwest (upright) or northwest-southeast (inverted) direction. In either case, the interference fringe should be well defined, as illustrated in Figure 7(b), and positioned in the center of the aperture.

        In some DIC microscope designs, the objective prism is fixed (de Sénarmont compensation), while in others the prism can be translated back and forth across the optical axis by means of a positioning screw mechanism in the slider frame. In the latter case, slowly twist the adjustment knob while observing the objective rear focal plane through the telescope or Bertrand lens. As the knob is turned, the interference fringe should move away from its central position to either the upper or lower half of the bright rear aperture. Alternatively, turning the polarizer in a de Sénarmont compensator will produce the same effect.

        Condenser DIC Prism Alignment - Remove the objective prism from the optical train, and swing the lowest aperture condenser prism (for use with the 10x objective) into position by rotating the condenser turret. The appropriate position is usually marked by the red or white 10 setting on the turret (or a similar code, such as L). Refocus the phase telescope or Bertrand lens to observe the interference fringe that appears in the objective rear focal plane. Once again, a single fringe should be present having the same orientation as the fringe produced by the objective prism (northeast-southwest for upright microscopes or northwest-southeast for inverted microscopes). The interference fringes for the condenser and objective prisms should appear almost identical and should have the same orientation along the shear axis.

        In order to clearly observe the condenser prism interference fringe using high-numerical aperture condensers designed for oil immersion, it may be necessary to remove the front lens assembly using the swing-lens control lever. If the fringe appearing in the objective aperture is not correctly positioned, it may be necessary to adjust the orientation or alignment of the condenser prism. In most cases, condenser prisms are assembled in a protective circular aluminum frame (Figure 3) with a notch or pin (or a lock-down screw) to ensure correct positioning within the condenser turret. Occasionally, a condenser prism can be forced into the turret without proper alignment, which will be apparent when the interference fringe is examined. If a condenser prism appears to be out of alignment, check with the microscope manufacturer for instructions on proper adjustment of the condenser DIC prisms.

        Specimen Observation - With the microscope aligned for Köhler illumination, the polarizer and analyzer crossed, and both prisms (objective and condenser) installed, place a thin transparent mounted specimen (such as a buccal mucosa epithelial cell preparation) on the stage. Adjust the microscope for maximum extinction, and focus the specimen while observing the procedure through the eyepieces in orthoscopic mode (no Bertrand lens or phase telescope). The image observed in the viewfield should appear very dark gray, almost black, at maximum extinction with bright highlights in regions having sharply defined thickness or refractive index gradients (for example, the cellular membrane and nucleus see Figure 8(b)). Spherical specimens with a high refractive index, such as immersion oil droplets, may even act as tiny lenses, and appear with a sharply defined interference fringe or band traversing the central region oriented in the same direction as the fringes are when observed in the objective rear aperture.

        While observing the focused specimen image, translate the objective DIC prism using the slider knob or rotate the polarizer (or analyzer) in a microscope equipped for de Sénarmont compensation. This action is termed introduction of bias retardation, and will translate the interference fringe bisecting the specimen along the shear axis and produce a corresponding change to specimen appearance. Shifting the prism in one direction (positive bias) will lighten specimen features at one edge while darkening the same features on the opposite edge and simultaneously lighten the background (see Figure 8(a)). In general, the specimen assumes a pseudo three-dimensional appearance with a shadow-cast effect oriented in the same direction as the shear axis. Moving the prism to the other side of the microscope optical axis (negative bias) will reverse the light and dark specimen regions (compare Figure 8(a) with Figure 8(c)).

        At maximum extinction with all DIC components properly installed and aligned, the objective rear aperture appears dark gray (almost black) and relatively uniform when observed with a phase telescope or Bertrand lens (Figure 7(c)). In most cases, the central region of the rear aperture appears jet black while some evidence of light appears in the four quadrants at the periphery. The extinction cross should generally appear quite similar to that observed with crossed polarizers alone, but usually is much darker and covers a larger region of the objective rear aperture. The bright areas surrounding the periphery (Figure 7(c)) result from an artifact that arises through partial depolarization of light at the surface of the polarizers and lens elements in the condenser and objective.

        Differential interference contrast images can be significantly improved by masking the bright regions at the periphery of the extinction cross in the objective rear aperture. This is accomplished by reducing the size of the condenser aperture diaphragm to eliminate the bright edges. In general, the objective rear aperture size should be reduced with the condenser diaphragm to approximately 75 or 80 percent of the full aperture. When the optical system is in perfect alignment, the extinction cross appears upright (see Figure 7) and can be observed to consist of two broad interference fringes, each shaped in a right angle and meeting at the center of the objective rear aperture (the fringes can also be visualized in orthoscopic mode in lower quality microscopes).

        On some microscopes, the condenser and objective prism positions can be adjusted to yield a more uniform fringe pattern, resulting in the central region of the aperture appearing darker and more uniform. This task is accomplished to loosening and rotating (or raising or lowering) the condenser prism or by uncrossing the polarizer and analyzer by a couple of degrees. Occasionally microscopes contain setscrews that allow for adjustment of the condenser and objective prisms, but models so equipped are becoming rare. As a final check on microscope alignment, adjust the condenser focus knob while examining the extinction pattern in the objective rear aperture to determine whether it can be improved. Note that a significant repositioning of the condenser may degrade optical performance by separating the overlap between conjugate interference planes of the objective and condenser DIC prisms.

        Adjusting the bias retardation by translating the objective prism, or rotating the polarizer in a de Sénarmont configuration, dramatically improves the image appearance (over that observed at maximum extinction) and increases contrast. This maneuver is essential for imaging specimens in differential interference contrast, and represents the last step in the adjustment of the microscope optical train. In many cases, a gradient of light appears across the entire field of view when observing DIC images. This occurs in addition to the presence of light and dark intensities at opposite edges of the specimen, and is due to a broad and indistinct field fringe artifact produced by the optical system. Microscopes having well-matched optical components maximize the size of the field fringe, which can become so broad and evenly distributed that the entire field appears a uniform medium gray color. In most cases, however, some evidence of the fringe remains, and the viewfield exhibits a shallow gradient of light intensity (medium to light or darker shades of gray) from one peripheral edge to the other. This artifact is inherent in a particular optical configuration and should be ignored when observing and collecting images of DIC specimens.

        Compensators in DIC Microscopy

        Specimen contrast can also be increased by introducing a retardation plate (or compensator) into the optical pathway in a DIC microscope. In general, a full-wave (also termed a first-order compensator) plate is inserted in an intermediate tube between the objective prism and the analyzer, although the plate can also be situated after the polarizer but before the condenser prism. These plates exhibit a retardation level of an entire wavelength at a specified value in the green region (usually near 550 nanometers), and result in the specimen displaying yellow and blue Newtonian interference colors along refractive index and thickness gradients. The background is rendered magenta due to the subtraction of green wavelengths from white light.

        In a de Sénarmont or a standard (translatable) Nomarski prism DIC microscope configuration, when the objective prism is positioned with the extinction interference fringe in the center of the optical pathway, a pattern similar to that displayed in Figure 7(b) is observed at the objective rear focal plane (provided the condenser prism is removed from the optical path). If a full-wave retardation plate is then placed between the objective prism and the analyzer, the interference pattern illustrated in Figure 9(a), which displays a spectrum of Newtonian interference colors, appears in the objective rear focal plane. Removing the objective prism from the light path and inserting a condenser prism yields the pattern presented in Figure 9(c). When both the objective and condenser prisms are present in the optical train and adjusted to the extinction position, a magenta color is visible in the objective rear focal plane (Figure 9(b)).

        Translating the objective Nomarski prism or rotating the polarizer in a de Sénarmont compensator configuration will shift the Newtonian interference pattern color illustrated in Figure 9(b). Introducing negative bias will shift the Newtonian colors to subtractive values (yellow), while shifting the prism to positive bias values will result in additive colors (blue). The colors produced by specimen gradients can be compared to a Michel-Levy reference chart in order to determine the magnitude of optical path differences.

        DIC Microscope Configurational Errors

        When a differential interference microscope is properly configured, the resulting images display a realism manifested in pseudo three-dimensionality through a shadow-cast effect that appears to originate from a highly oblique angle (see Figure 10(a)). However, even slight alignment errors can result in a deterioration of minute specimen detail, and serious mistakes in configuration can yield a dramatic loss of contrast that renders the image useless. The most common errors in DIC microscope configuration result from polarizers that are not crossed, a mismatch between the objective and condenser prisms, a missing condenser DIC prism, or incorrect setting of the condenser iris aperture. Other problems can arise, especially if the condenser and/or objectives are not strain free, but these mistakes are rare if the microscope is designed specifically for imaging specimens in DIC illumination.

        Ensuring that the polarizers are crossed and positioned in the correct orientation was discussed previously in the section on microscope alignment. Occasionally, either the polarizer or analyzer can become accidentally rotated out of position by brushing against the thumbwheel or careless use of the instrument. As a result, overall specimen contrast is reduced, but many features often remain visible. This type of mistake can be eliminated by utilization of a fixed polarizer and analyzer or by ensuring that the locking knob is set to suspend polarizer rotation. Most polarizers housed in rotating mounts contain marks that indicate the position of the transmission azimuth. A careful check of the polarizer and analyzer orientation prior to commencing DIC observations will avoid errors with these components.

        Accidental mismatch between the objective and condenser prisms is another common source of errors in differential interference contrast microscopy. Because of the shear angle discrepancies between prisms not designed to work together, the resulting image exhibits extremely poor contrast (see Figure 10(c), which cannot be corrected by translating the objective prism or rotating the polarizer in a de Sénarmont compensation microscope. The condenser turret is usually marked with a code that identifies the prism placed into the optical pathway. Inspection of the exterior code order to ensure that condenser prisms are positioned in the correct slot will help to avoid this type of error. Also, the prism code inscribed on the objective barrel should match the code for the condenser prism in use. In general, prism mismatches occur when the magnification is changed, but the condenser turret is not rotated to insert a matching prism into the optical pathway. Occasionally, a condenser prism may be missing from the condenser or the condenser turret rotated into a position that does contain a prism (for example, the brightfield position see Figure 3). The result is an image similar to that illustrated in Figure 10(c), which lacks contrast and does not display the high resolution obtainable with differential interference contrast.

        The condenser iris diaphragm is typically employed to adjust depth of field and contrast for images observed in the eyepieces or recorded digitally and on film. In differential interference contrast microscopy, wide aperture sizes are useful for optical sectioning experiments, where a shallow depth of field is required, but high resolution is also necessary. Often, the compromise between maximum contrast afforded by using smaller apertures is offset by the elimination of interfering specimen features located away from the plane of focus. If the condenser aperture is not opened sufficiently (to about three-quarters to four-fifths of the objective aperture), diffraction artifacts can obscure important specimen detail and seriously degrade image appearance (Figure 10(b)). In general, diffraction effects can be avoided by maintaining the condenser iris diaphragm size between 80 to 90 percent of the objective aperture.

        Additional errors can occur due to spherical aberration when using thick specimens immersed in water, or when the glass microscope slide, culture dish, or coverslip is too thick. These mistakes are manifested by images that are not sharply focused and lack the shadowed relief that is characteristic of DIC images. Correction collars on high magnification dry objectives can often be adjusted to correct for spherical aberration, but specimens that are too thick must be replaced with thinner sections. When examining cells in culture, do not use DIC optical systems with culture vessels made from injection-molded polymers. These materials display stress birefringence and will produce confusing images. Instead, use glass vessels designed especially for DIC microscopy, which are available from several manufacturers. In general, careful attention should be paid to selection of high-quality glass microscope slides and coverslips manufactured to precise tolerances, and the glass should be thoroughly cleaned and dried prior to preparation of specimens.

        Illumination Sources for DIC Microscopy

        At lower magnifications (10x through 40x), a 50-watt quartz halogen lamp can provide enough light for satisfactory observation and recording of images in DIC. However, at the highest magnifications (60x and 100x), at least a 100-watt tungsten-halogen light source is recommended. The illumination intensity required by the light source is dependent upon the transmission percentage of the polarizer and analyzer, and many older DIC microscopes are equipped with polarizing elements that have low transmission values (20 percent or less). However, later model microscopes are often equipped with high transmission polarizers (greater than 30 percent), which enable a higher flux density to pass through the optical system, providing adequate light for observation and imaging at the highest magnifications.

        Although coarse structural detail, such as the edges of cells and larger intracellular components (nuclei) can be easily visualized with a high-intensity tungsten-halide lamp, in order to observe the finest structural details, very intense illumination having a narrow bandwidth is essential. Mercury arc-discharge lamps feature a high-energy peak in the central portion of the visible spectrum at 546 nanometers (green), which can be further refined by passage through a narrow bandwidth (10 nanometer) interference filter centered at 546 nanometers. In addition, a light scrambler can be employed to enhance the narrow bandwidth illumination to achieve the highest possible resolution. Note that the objective aperture should be fully illuminated, regardless of the light source, for the best results.

        The correct placement and orientation of optical components in a differential interference contrast microscope is critical to optimum performance, regardless of the target magnification and resolution. Improper adjustment of even a single component can result in serious image degradation and upset the shadow-cast effect and resolution of the instrument. The two compound prisms and their associated polarizers in DIC microscopy are mirror image pairs. Light waves passing through the initial polarizer and condenser prism are imaged onto the objective prism and analyzer to produce the final image. All four of these components must be in the proper orientation in order for DIC to function properly. Contrast is generated either by altering the lateral position of the objective prism, or by rotating the polarizer in de Sénarmont compensation instruments. In this manner, the optical path difference between wavefronts is increased or decreased to produce contrast in the specimen.

        It should be noted that contrast in DIC is a function of gradients in the specimen optical path length, which are rendered in pseudo three-dimensional relief when the microscope is properly adjusted. Because contrast arises from a combination of refractive index fluctuations and/or thickness variations, apparent gradients observed with the technique can correspond to real changes in specimen topography or may be a function, for example, of localized protein concentration gradients. Only independent knowledge of the structural properties relating to the specimen can help determine the absolute nature of contrast effects in DIC microscopy.

        In addition, even when it is determined that shadow-cast effects can truly be ascribed to height changes in the specimen, there is no inherent mechanism in DIC to indicate which features represent plateaus or valleys at a particular setting of the objective prism. A qualitative solution to the problem is to locate a known specimen characteristic, such as a scratch in the glass, and observe how the feature responds to bias retardation as the objective prism is translated across the optical axis. Integral specimen features having a similar response will be either valleys or plateaus, while those with opposite responses will have a reverse orientation.

        When examining unknown specimens with differential interference contrast, the microscopist should be alert to interference colors arising at the edges, which are normally featureless and exhibit either a very light or dark color, depending upon orientation. The presence of interference colors indicates that the specimen may be birefringent and, consequently, not a good candidate for DIC observation. This fact can be confirmed by examining the specimen under crossed polarized illumination with both the objective and condenser DIC prisms removed from the optical pathway.

        Among the limitations imposed on differential interference contrast microscopy are the expensive birefringent Nomarski or Wollaston prisms necessary to perform the technique. These components are far more expensive than those required for phase contrast or Hoffman modulation contrast microscopy, which may serve as alternative techniques, especially when observing living cells in plastic vessels. In addition, very thin or scattered specimens often produce better images having more contrast when phase contrast is used instead of DIC. Older apochromatic objectives may not be suitable for DIC observation because the objectives themselves can significantly affect polarized light. The microscopist should check with the manufacturer before purchasing high-quality apochromatic objectives in conjunction with DIC optical components.

        Contributing Authors

        Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

        Edward D. Salmon - Department of Cell Biology, The University of North Carolina, Chapel Hill, North Carolina 27599.

        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.


        Viruses

        Viruses are intracellular parasites that cannot replicate on their own. They reproduce by infecting host cells and usurping the cellular machinery to produce more virus particles. In their simplest forms, viruses consist only of genomic nucleic acid (either DNA or RNA) surrounded by a protein coat (Figure 1.42). Viruses are important in molecular and cellular biology because they provide simple systems that can be used to investigate the functions of cells. Because virus replication depends on the metabolism of the infected cells, studies of viruses have revealed many fundamental aspects of cell biology. Studies of bacterial viruses contributed substantially to our understanding of the basic mechanisms of molecular genetics, and experiments with a plant virus (tobacco mosaic virus) first demonstrated the genetic potential of RNA. Animal viruses have provided particularly sensitive probes for investigations of various activities of eukaryotic cells.

        Figure 1.42

        Structure of an animal virus. (A) Papillomavirus particles contain a small circular DNA molecule enclosed in a protein coat (the capsid). (B) Electron micrograph of human papillomavirus particles. Artificial color has been added. (B, Alfred Pasieka/Science (more. )

        The rapid growth and small genome size of bacteria make them excellent subjects for experiments in molecular biology, and bacterial viruses (bacteriophages) have simplified the study of bacterial genetics even further. One of the most important bacteriophages is T4, which infects and replicates in E. coli. Infection with a single particle of T4 leads to the formation of approximately 200 progeny virus particles in 20 to 30 minutes. The initially infected cell then bursts (lyses), releasing progeny virus particles into the medium, where they can infect new cells. In a culture of bacteria growing on agar medium, the replication of T4 leads to the formation of a clear area of lysed cells (a plaque) in the lawn of bacteria (Figure 1.43). Just as infectious virus particles are easy to grow and assay, viral mutants𠅏or example, viruses that will grow in one strain of E. coli but not another𠅊re easy to isolate. Thus, T4 is manipulated even more readily than E. coli for studies of molecular genetics. Moreover, the genome of T4 is 20 times smaller than that of E. coli—approximately 0.2 million base pairs𠅏urther facilitating genetic analysis. Some other bacteriophages have even smaller genomes—the simplest consisting of RNA molecules of only about 3600 nucleotides. Bacterial viruses have thus provided extremely facile experimental systems for molecular genetics. Studies of these viruses are largely what have led to the elucidation of many fundamental principles of molecular biology.

        Figure 1.43

        Bacteriophage plaques. T4 plaques are visible on a lawn of E. coli. Each plaque arises by the replication of a single virus particle. (E. C. S. Chen/ Visuals Unlimited.)

        Because of the increased complexity of the animal cell genome, viruses have been even more important in studies of animal cells than in studies of bacteria. Many animal viruses replicate and can be assayed by plaque formation in cell cultures, much as bacteriophages can. Moreover, the genomes of animal viruses are similar in complexity to those of bacterial viruses (ranging from approximately 3000 to 300,000 base pairs), so animal viruses are far more manageable than are their host cells.

        There are many diverse animal viruses, each containing either DNA or RNA as their genetic material (Table 1.3). One family of animal viruses—the retroviruses𠅌ontain RNA genomes in their virus particles but synthesize a DNA copy of their genome in infected cells. These viruses provide a good example of the importance of viruses as models, because studies of the retroviruses are what first demonstrated the synthesis of DNA from RNA templates𠅊 fundamental mode of genetic information transfer now known to occur in both prokaryotic and eukaryotic cells. Other examples in which animal viruses have provided important models for investigations of their host cells include studies of DNA replication, transcription, RNA processing, and protein transport and secretion.

        Table 1.3

        Examples of Animal Viruses.

        It is particularly noteworthy that infection by some animal viruses, rather than killing the host cell, converts a normal cell into a cancer cell. Studies of such cancer-causing viruses, first described by Peyton Rous in 1911, not only have provided the basis for our current understanding of cancer at the level of cell and molecular biology, but also have led to the elucidation of many of the molecular mechanisms that control animal cell growth and differentiation.


        Watch the video: 10. Widefield Microscopy Training: Differential Interference Contrast DIC Imaging (May 2022).