Microscopy Essay, Research Paper

Perhaps no single instrument has advanced man’s understanding of the surrounding world more than the microscope. Scientific discoveries made through microscopic techniques are too numerous to list. The microscope revolutionized the study of biology, medicine, and many other fields of scientific research.

Basic Principles

Light microscopes use convex (converging) lenses. The way a lens magnifies an object depends on where its placement relative to the focal point of the lens. If the object is further away than the focal point the result is a real image, one that can be photographed or projected (Rawlins, 1992). The image will be upside down if viewed with a single lens but right side up for a compound microscope. A virtual image is created when an object is placed within the focal point of the lens. The light rays passing through the lens do not converge but diverge instead. By using an additional convex lens the image can be formed (rightside up) which is said to exist “at infinity” (Lacey, 1989). This means that the image can be seen with a relaxed eye, as if viewing a distant object.

A compound microscope is made of two convex lenses. The objective lens is the lens that is closest to the specimen. It is essentially the information gathering lens of an optical system. Therefore, it is regard as the most important lens of the microscope. There are many different types of objective lens. The most common and inexpensive is the achromat. This lens is usually found on student microscopes. It is corrected for spherical aberration for only green light. Chromatic aberration is corrected in only two colors. The apochomat objective is far superior and generally very expensive. Chromatic aberration is corrected for all three colors and is spherically corrected for two colors. These objectives quite often will require a special compensating eyepiece(Rawlins, 1992).

Magnification

The magnifying power of a microscope is determined by multiplying the magnification of the ocular (eyepiece) and the objective lens. For example, a low-power objective might have a magnification of 4x and a high-power oil-immersion objective 100x. If each is used with an ocular of 10x magnifying power, magnifications of 40x and 1,000x are obtained.

The eyepiece is basically a projection lens system. There are three types generally used in light microscopy. The most common is the Huygenian type. This eyepiece is used with low and medium magnification and is designed to project the image into a human eye. The second type of eyepiece is the compensating eyepiece and is generally used with apochromate or flat field objectives. The third type is the photo eyepiece, designed to project a corrected image onto film plane in a camera. Photo eyepieces are generally considered the finest of eyepieces(Lacey, 1989). All eyepiece will have a relative magnification written on the side of the barrel. They range in magnification from 2.5X to 15X with the lower magnifications used with the photo eyepiece.

The objective lens is composed of several lens elements that form an enlarged real image of the object being examined. The real image formed by the objective lens lies at the focal point of the ocular lens. Thus, the observer looking through the ocular lens sees an enlarged virtual image of the real image. The total magnification of a compound microscope is determined by the focal lengths of the two lens systems and can be more than 2000 times.

The smallest object that can be seen in an optical microscope is limited by the wave character of light to a size the order of one light wavelength. Under optimal conditions, using an oil-immersion lens, in which a drop of oil is placed on the slide and the lens is dipped into the drop, objects as small as 200 nm can be resolved. This limit is reached with a magnification of about 1,000x. Additional magnification over the minimum is called empty magnification because the image is only coarsened and no additional information can be obtained(Rawlins, 1992).

Resolution

Resolution is the ability to see two objects as separate. If two objects are too close together the light coming from them will be focused on the same cell of the retina and they will appear as a single object. A human eye can resolve two objects as close together as 150mm. Lenses can increase resolution by increasing the angle of light between the two objects that reaches the retina. Resolution is limited by the light gathering power of the lens (numerical aperture) and the wavelength of the light used. Numerical aperture is determined by the formula:

NA=h*sina

Where h is the refractive index of the medium between the specimen and the lens, and a is the angle between the cone of light and vertical(Rawlins, 1992). The resolution will be expressed in the same units as the wavelength of the light. The maximum theoretical value for sin a is 1. Therefore the highest theoretical value of the numerical aperture for oil immersion viewing (refractive index 1.515) is 1.515. However the highest value that can be achieved under practical circumstances is 1.4. The practical resolution will always be less due to optical aberrations.

Ernst Abbe was able to derive an expression for resolution by optical geometry(Lacey, 1989). The Abbe equation is based on the size of the lens that will capture the light. The relationship between wavelength and resolution is:

resolution= 0.61* l / NA

For example, if green light is used (l=500nm) with a numerical aperture of 1.4, the closest together that two objects could be resolved as separate would be 218nm. Electron microscopes can resolve objects much closer than this because the l is much smaller than for visible light.

Numerical Aperture

The numerical aperture (NA) is basically a value that describes the quality of a lens. It is derived from the size of the lens, its working distance and the index of refraction. All quality objective lens will state the numerical aperture on the side of the barrel. A good rule of thumb is that the effective magnification of an objective is its numerical aperture times 1000(Lacey, 1989). So a 40x objective that has a NA of 0.65 has an effective magnification of 650 times. Magnification beyond this will not give any further information about the specimen.

Depth of Field

Numerical aperture is also the single most important parameter that determines depth of field. Depth of field is the area in front of and behind the specimen that will be in acceptable focus. For example, when you take a photograph of a close up of a person the background will often be out of focus. When a lens is at its full aperture opening (Figure1) the depth of field is decreased.

Figure 1: Depth of field with full aperture

On the right side focus is a vertical line representing the specimen plane. The horizontal line shows the range of acceptable focus. The criteria for acceptable focus is ultimately dependent on the circle of minimum confusion, the summation of all the optical aberrations(Rawlins, 1992). In a practical sense the acceptable focus is dependent on effective magnification. The higher you magnification of an object the more critical the focus.

When the numerical aperture of the lens is stopped down by an aperture (Figure 2) there is a decrease in the angle of acceptance. Since the rays of light are now at a shallower angle, the range of focus is increased. The focal length of a lens is also a factor in controlling depth of field. Since the angle of acceptance is dependent on the focal length, which in turn determines the numerical aperture(Lacey, 1989).

Figure 2: Depth of field increases with a smaller aperture

A large lens with short focal length at high magnification will have a very short depth of field. A small lens with long focal length and low magnifications will have a much greater depth of field

Depth of focus

The range of acceptable focus for the image is called depth of focus. It is essentially the same as depth of field except that with higher magnification depth of field decreases but the depth of focus increases.

Contrast

Contrast is the ratio between the dark and the light. A high contrast picture will have only two shades, black and white. The more shades there are, the less contrast. More contrast does not necessarily mean more information. Optically speaking, contrast is necessary since it is possible to generate an image of high resolution but it is the contrast that lets you see it(Lacey, 1989). In standard bright field microscopy contrast and resolution are mutually exclusive. The result is if you have high contrast you will have poor resolution. In bright field microscopy, absorption contrast is used. The light is literally absorbed by pigments in the specimen. The result is less light is transmitted to the eye so the specimen appears dark. If the pigments absorb only a specific wavelength of light the specimen will appear the complimentary color. The use of stains can dramatically increase the absorption contrast of a specimen. There are other types of microscopes that use more exotic means to generate contrast, such as phase contrast, dark field, differential interference contrast. Diffraction contrast occurs when light hitting the edge of the specimen bends and is diffracted out of the optical path. This is the mechanism used for dark field and stop contrast microscopy.

Other Microscope Parts

Illumination Source

The higher a microscope’s magnification, the more light will be required. The illumination source should also be at a wavelength that will facilitate the interaction with the specimen. All microscopes fall into either of two categories, diascopic and episcopic (Figure 3), based on how the specimen is illuminated(Lacey, 1989). In the typical compound microscope the light passes through the specimen and is collected by the image forming optics. This is called diascopic illumination. Dissecting (stereo) microscopes have episcopic illumination for use with opaque specimen. The light is reflected onto the specimen and then into the objective lens. Dissecting scopes also have diascopic illumination for use with a transparent specimen or to enhance edge contrast (Rawlins, 1992).

Figure 3: Diascopic Illumination vs Episcopic Illumination

Condensers

The substage condenser of a microscope is design to focus the light onto the specimen and fill the numerical aperture of the objective. The most common type of condenser, the Abbe condenser, is not corrected for optical aberrations. The achromatic condenser is corrected for both spherical and chromatic aberrations. Both types of condenser have their numerical aperture printed on the side. The NA should be of equal or greater value then that of the objective. If it is not, the full resolution of the objective will not be utilized. Most substage condensers can use immersion oil like that of the objectives to achieve their full NA (Rawlins, 1992). However, this is rarely done except in photomicroscopy.

Specimen Preparation

Specimens for microscopy are mounted on glass slides and covered by a thin glass coverslip. Fine particulate materials such as powders and blood smears can be examined without further preparation, but most specimens are too thick to be seen in this way. Specimens are prepared as sections (slices 100 micrometers thick or less). To prepare for sectioning, a biological specimen is first preserved and hardened by infiltration of a chemical fixative such as formaldehyde. The fixed material is dehydrated by a series of solvents, embedded in a wax or other medium for cutting, and mounted in a microtome. A microtome holds the embedded tissue at a chosen orientation for cutting to a thin, reproducible thickness by an extremely sharp knife edge. The sections are collected on slides and may be stained with dyes to reveal various features through absorption contrast.

Research

The paper I chose to review was; The effects of pH on arbuscular mycorrhiza (field observations on the long term-liming liming experiments at Rothamsted and Woburn). The paper measured the percentage of colonization of the roots of spring oats (Avena sativa) and maincrop potatoes (Solanum tuberosum) by arbuscular mycorrhizal fungi(AMF). Arbuscular mycorrhizal fungi invest plant roots penetrating into the root cortex. AMF produce hyphae, filamentous fungal branches, which extend much farther into the surrounding soil than the host’s roots. The increased absorption area has been shown to increase nutrient uptake and drought resistance of the plant partner (Schenk,1982). In return, the fungal partner is supplied with sugars from the plant, that act as its sole carbon source. Arbuscules, structures within the plant roots, are the sites of this two-way exchange.

AMF have been shown to increase the host plant’s defenses against heavy metal concentrations in the soil, (Wiesenhorn, 1994). Increasing acidity in soils may increase the availability of aluminum and manganese in the soil solution. This study attempted to determine whether soil acidity affected AMF colonization, either directly or by selection of acid tolerant species within the original population. Previous studies (Robson & Abbott, 1989) have shown that spore germination of AMF spores is affected by pH and heavy metal concentration, with spores of different species having differing pH optima.

The chosen sites, Rothamsted Experimental Station and Woburn, have a series of plots that have been maintained at four different pH values for 22 years. Preliminary studies have shown that soil pH had no effect on the percentage of roots colonized in oat and potato but did affect the species of the colonizing AMF fungi (Wang et al., 1985). Oat and potato were chosen for their ability to tolerate a wide range of soil pH. Three soil cores (7.5 x 30cm) were taken from each site. Roots were washed from the cores and cut into lengths of 1cm from which a subsample was taken. Roots were cleared and stained and the percentage of AMF colonization was assessed using the gridline intersect method of Giovannetti & Mosse (1980). AMF spores were extracted from fresh soil by wet sieving and decanting. Spores over 50mm were counted on a nematode-cyst counting dish.

The clearing and staining technique used was that of Phillips and Hayman (1970). This procedure was a major breakthrough in AMF research because it was specifically adapted to mycorrhizal fungi and did not require the embedding and sectioning that was previously used. There are drawbacks to this procedure including the use of phenols or saturated chloral hydrate (Schenk, 1982). The fumes of both of these are hazardous even at room temperature, and the procedure required them to be heated. Newer procedures can adequately stain AMF infection using only lactic acid instead of phenols, (Kormanik, 1980). The gridline intersect method (Giovannetti and Mosse, 1980) can be used to estimate both the proportion of root length colonized and the total root length of the sample. The procedure involves spreading the washed root sample in a petri dish. The dish is placed on a grid of 1.27cm squares and viewed through a dissecting microscope. Vertical and horizontal gridlines are scanned and presence or absence of colonization at each root/line intersection is recorded. High accuracy of percentage of colonization has been determined if at least 100 intersections are tallied. For estimates of total rootlength all intersections must be recorded. The total number of root/gridline intersections will represent the total root length in centimeters. Many other procedures have been developed to assess AMF colonization. The magnified intersect method (McGonigle et al., 1990) involves scanning roots at higher power (200x) using an eyepiece with two perpendicular crosshairs. This technique has been reported to give a more objective and accurate representation of AMF colonization.

Soil pH effected the crop yields of spring oats and potato. The percentage of AMF root colonization could account for these differences because a change in percentage can result from changes in root growth rate or fungal growth rate. The highest percentage of colonization was observed in soils with a pH of 6.5, but changes within the range 5.5-7.5 were small. On the most acidic plots (pH 4.5) colonization was reduced by half over the pH 7.5 plot. The percentage of colonization increased only slightly over time. Overall, available phosphorous seemed to affect the percentage of colonization more than soil pH. At sites with a greater amount of available phosphorus root colonization was strongly suppressed. The results were similar for potato except that the lowest percentage of colonization was observed at the pH 7.5 site at Woburn but at the pH 4.5 site at Rothamsted. This may be the result of the difference in soil chemistry of the two sites. Rothamsted has a greater concentration of has a higher concentration of oxides of aluminum, iron and manganese in the soil, which would become more available at low pH. This could account for the suppression of colonization. However, a similar effect was not observed in oats.

Because of the similarity of colonization rates across a wide range of pH, the species of colonizing fungi was of great interest. Spores were separated from the soil and identified, but this does not give a clear indication of the amount of colonization of roots by these species. The number of spores of a particular species in the soil and the percentage of colonization of a plants roots are not necessarily related. Non-mycorrhizal species invasion of the test plant roots made identification of species difficult. However only the fine endophyte species were found in the acidic soils and only the course type were found at pH 7.5. No resting spores of the course endophytes were found in any soil at pH 4.5, but since spores less than 50mm were not sampled and some species of AMF are known to have smaller spores at maturity, species of course endophyte may have been present. Soil from the other pH sites was found to have up to nine different species, six of which were identified. Three species (Glomus caledonium, G. albium and G. etunicatum) occurred in almost all treatments at both sites. The shift in species across pH appeared to have a very limited affect on the percentage of colonization. The authors suggest that the uniformity of colonization suggests mechanisms within the host plant rather than the concentrations of inoculum of the soil, affect colonization. Natural selection may alter species composition in response to long-term changes of the soil environment, while leaving concentrations of inoculum relatively unchanged.

Suggested Further Research

The arbuscles of many species of AMF have been shown to exhibit autofluorescence, absorbing at 455-490 nm and transmitting at 520-560 nm (Ames, 1982). If the researchers had access to this technology, the same roots used for AMF colonization assessment could also have been used to inoculate pot cultures for fungal species determination. Because no staining is needed, fungal spore viability is not compromised. After the AMF colonization percentage is determined, the roots are surface sterilized and planted with a similar host species in sterilized soil. Wet sieving and decanting the soil will yield only the spores of the species that colonized the original plant of interest (Dodd, personal communication). Fitting plants to soil through the determination of a suitable fungal partner could improve plant viability in stressful environments. Chosing the correct fungal partner for sropp plants could save time, money and environalso reduce the need well as decreasing the need for applied fertilizers.

References

Lacey, A.J. (1989) Light Microscopy in Biology. IRL Press, Oxford UK

Rawlins, D. (1992) Light Microscopy .BIOS Scientific Publishers Ltd, Oxford, UK.

Schenck. N. C. (1982) Methods and Principles of Mycorrhizal Research. American Phytopathologiocal Society, St. Paul, Minn.

Ames, R., E. Ingham & C. Reid (1982) Ultraviolet induced autofluorescents of arbuscular mycorrhizal root infections: An alternative to clearing and staining methods for assay infections. Canadian Journal of Microbiology. 28:485-488

Giovennetti, M., & B. Mosse (1980) An evaluation of techniques for measuring VAM infection in roots. New Phytol. 71:287-295.

Kormanik, P., W. Bryan & R. Schultz (1980) Procedures and equipment for staining large numbers of plant roots for mycorrhizal assay. Can J. Microbiol. 26:536-538

McGonigle, T., M. Miller, D. Evans, G. Fairchild and J. Swain (1990) A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 33:115

Mosse, B., D. Stribley & F. LeTacon (1981) Ecology of mycorrhiza and mycorrhizal fungi. Advances in Microbial Research. 5:137-210.

Phillips, J., & D. Hayman (1970) Improved procedures for clearing and staining parasitic and VAM fungi for rapid assessment of infection. Transactions of the British Mycological Society. 55:158-161

Wang, G., D. Stribley, P. Tinker and C. Walker (1993) Effects of pH in arbuscular mycorrhiza, field observations on the long-term liming experiments at Rothamsted and Woburn. New Phytologist 124:3

Dr.J.C.Dodd, Director, International Institute of Biotechnology MIRCEN,(& Dept. of Biosciences),

University of Kent. Canterbury, UK.