LIGHT MICROSCOPY

MICROSCOPY: Histroy

The fascinating world of microorganisms would have remained unknown had the microscope not been invented. Roger Bacon (1267) described a lens for the first time. However, his observation neither was nor pursued immediately thereafter. In 1950, Hans and Zacchrius Jensen constructed a crude type of operational microscope (10-30 times magnification) by placing two lenses together which permitted them to see minute objects. In 1609-1610, Galileo Galilei made the first simple microscope with a focussing device called 'occiale' and observed the water flea through his microscope.

In 1617-1619, the first double lens microscope with a single convex objective and ocular appeared, the inventor of which was thought to be the physicist C. Drebbel. This microscope was used to study the cells, plant and animal tissue and also the minute living organisms. Till then, the name microscope had not been given to this device; the name 'microscope' was first proposed by Faber (or Fabri) in 1625.

The credit of developing a compound microscope with multiple lenses goes to Robert Hooke (1665) of England. It was only after 1670 that a cloth merchant of Deflt (Holland), Antony van Leeuvenhoek (1632-1723), started his hobby of making microscopes and in 1674 he discovered the fascinating microbial world through his microscope (50-300 times magnification).Considerable progress was made in improving the microscope in nineteenth century. The introduction of oil-immersion by Amici in 1869, substage condenser by Abbe in 1872, apochromatic objectives with suitable eyepiece by Abbe and Zeiss in 1886 were landmarks in the improvement of compound microscope in the nineteenth century.

LIGHT MICROSCOPY

1. Compound (Bright-Field or Light) Microscope

A compound microscope is the primary tool in microbiology. Therefore, a clear understanding of structure, use and manipulations of a compound microscope is a must for all students of micro­biology.

Parts of Monocular Compound Microscope

The essential parts of usually used monocular compound microscope are the following:

(i) Lense. The eyepiece with different magnification (5 20 times). It has field lens towards the object and eye-lens close to the observer's eye. The objectives are generally with three different magnifications viz., low power (10 X), high power (40-45 X) and oil-immersion (90-100 X). The focal length of these are 16 mm, 4 mm, and 1.8-2.0 mm respectively. These objectives are mounted on a revolving nosepiece for convenience. The eyepiece and objectives are fitted at the two ends of a hollow tube called the 'body tube".

The compound microscope showing its various parts

(ii) Adjustment of objective lens. In some microscopes coarse and fine focussing adjustment knobs are provided in order to lower or raise the body tube with lenses for rendering image clear. This is done by rotation of the knobs. The coarse adjustment is meant to bring to object into vision whereas the fine adjustment is used for focussing finer details.

(iii) Stage. The object to be: observed is kept on a glass slide and placed on the stage. It may have clips to keep the slide in desired position or a mechanical stage for horizontal movement of the object. In some microscopes the stage may be raised or lowered with coarse and fine adjustment for focussing the object.

(iv) Mirror. The mirror reflects light which is transmitted through the object for observing it. The mirror has two planes, one concave and the other plane. When natural light is available the plane mirror may be used for reflection of light because concave mirror would form window images. However, with artificial illumination, the concave mirror is necessary for higher magnifications whereas for lower, the plane mirror may be used.

(v) Substage diaphragm. This is meant to control the amount of light transmitted through the object.

(vi) Substage condenser. The substage condenser consists of convex lenses which concentrate and intensify the light reflected by the mirror. With objectives of magnifications exceeding 10 X, the use of condenser becomes necessary for narrowing the core of transmitted light which would fill the smaller aperture of the objective. The condensers usually employed are called' Abbe' condensers and these are used with plane mirrors.

Use
Having calibrated the eyepiece scale for all the objective lenses on the microscope, one can use it to measure the dimensions of cellular and subcellular structures, e.g., bacterial cells, fungal spores, onion epidermal cells etc.

2. Dark-Field Microscope

Dark-field microscopy permits the detection of unstained small biological objects which otherwise provide insufficient contrast. In a dark-field microscope the normal condenser of a light microscope is replaced with a dark-field condenser that does not permit light to be transmitted directly through the object and thus through the objective lens.

The dark-field condenser focuses light on the specimen at an oblique angle in such a way that the light does not impinge on the object and, does not enter the objective lens. Thus, only light that reflects-off the object is seen, and in the absence of the object the entire field appears dark. This microscope helps viewing even small bacteria and large viruses but it is not helpful in distinguishing the internal structure of any bacterium being viewed.

Dark-field microscopy showing the path of light

3. Phase Contrast Microscope

This microscope was developed by Fritz Zernikes (1935), a Dutch physicist who was awarded Nobel Prize in 1953 for this contribution. It is a conventional light microscope fitted with a phase-contrast, objective and a phase-contrast condenser . Phase contrast microscope is based on the fact that the rate at which light travels through objects is inversely related to their refractive indices.

Thus, the light passing through one object into another object of a slightly different refractive index undergoes a change in phase. These differences in phase are translated into variation in brightness of the objects and hence the objects differing even slightly in refractive index are viewed by the eye. Phase contrast microscope helps viewing living unstained structures of microbial cells. Unlike interference contrast microscope, the phase contrast microscope relies upon a single beam of light.

Phase Contrast Microscope Showing Path of Light

These microscopes produce high contrast images of unstained, transparent specimens in what appear to be three-dimensional. The three-dimensional image is produced because the two beams of light travelling very close to each other through the specimen produce a stereoscopic effect. Although these microscopes are very useful for qualitative observations of unstained cells, they do not produce clear images if the object viewed is very thin.

Fluorescence Microscope

Fluorescence microscope developed by Coons (1945), is that in which a specimen stained with fluorescent dye is viewed. A fluorescent substance is that which absorbs light of one wavelength (the excitation wavelength) and emits light at a different wavelength (the emission wavelength). For example, when the fluorescent dye "fluorescein isothiocynate" is illuminated with blue light, it emits green light. Fluorescent microscopes are equipped with excitation filters. It permits the selection of the wavelength used to illuminate the specimen and barrier filters that prevent all but the emission wavelength from reaching the ocular lens. If the dye is exposed to ultraviolet light, the emitted light to be viewed must be in the visible range.

Fluorescent microscopy has become important in microbiology as the fluorescent dyes can be linked with antibodies. When such antibodies combine with specific antigen, they become fluorescent. Thus, this technique makes possible to detect specially the cells of a particular type of bacterium in a mixed microbial population.

Fluorescence microscope showing path of light

5. Interference Contrast Microscope

This microscope (developed by Merton et at. 1947) relies upon the two beams of light illuminating the specimen and they combine after passing the specimen. Nomarski Differential Interference Contrast (NDIC) Microscopes are the most frequently used interference microscopes by the microbiologists.

Nomarski interference micrscope showing the path of Light

6. Ultraviolet (UV) Microscope

As we know that the resolving power of a light microscope is related to the wavelength of the light used; longer the wavelength lower the resolving power. Therefore, resolution can be improved by reducing the wavelength of the light. The UV microscope has this advantage.

Since glass is opaque to ultraviolet light, the lens system must be made of appropriate quality quartz and the microscopes should have filters to eliminate ultraviolet light from reaching the eyes. This microscopy is complicated and expensive thus a modification known as fluorescence microscopy has come into use.

Comparison of Various Types of Microscopes

Type of microscope	Maximum magnification	Resolution	Remar Remarks
Compound (Bright field or light)	1500 ×	100-200 nm	Extensively used to view microorganisms; objects are required to be stained for visualization.
Phase contrast	1500 ×	100-200 nm	Used for the visualization of living micro­organisms; staining not required.
Interference	1500 ×	100-200 nm	Used of view microbial structures; sharp, multicoloured, three-dimensional images are obtained
Ultraviolet	1500 ×	100 nm	Provides improved resolution over compound microscope. Replaced by fluorescence microscopes.
Fluorescence	1500 ×	100-200 nm	Used in many diagnostic procedures for identifying microorganisma; fluorescent dye used for staining.
Dark-field	1500 ×	100-200 nm	Used to view the microbes particularly with characteristic morphology; staining not required; object appears bright on a dark background.

Method for Studying Microbes with a Compound Microscope

Two -methods are generally used, 'wet method' and 'dry' and 'fix method'.
A. Wet Method. There are two primary methods generally used for studying microorganisms in wet conditions

(i) wet mount method, and

(ii) hanging drop method.

(i) Wet mount method. It is the most widely used method A drop of fluid containing microorganisms to be-examined put on a glass -slide and a coverslip made of thin glass is placed on it. The fluid spreads out in a thin layer between coverslip and slide. The mount is now examined under the microscope. For higher magnifications (e.g. with 100 X objective) the oil-immersion technique is employed.

A drop of immersion oil is put between the objective lens and cover slip before the microorganisms are examined under the microscope. The immersion oil fills the space between the specimen and the objective lens and thus replaces the air present between the specimen and the objective lens. The result is that the numerical aperture (NA) is improved and the level of magnification is increased.

The wet mount

(ii) Hanging drop method. It is used to observe the motility, germination, or fission of microorganisms. In this method a cavity slide which has a circular concavity in the centre is used.The periphery of the concavity on the cavity-slide is smeared with vaseline. A drop of liquid microbial culture is placed in the centre of the cover glass if it is a liquid culture.

If the culture is solid, it is mixed with a drop of distilled water before placing on the cover glass. The cover glass is inverted over the concavity so that the drop hangs freely and the edge of cover glass adheres tightly to the vaseline coated periphery of the concavity. The microorganisms present in the hanging drop are now observed under the microscope.

The hanging Drop Method

B. Dry and fix method. Microorganisms, particularly bacteria, being too small need their permanent preparation be made by drying and fixing them on clean slide with or without staining. For preparing a dry mount, a drop of distilled water with a small amount or culture is spread as a thin smear on a clean slide. The smear is allowed to dry and it is then 'fixed' by passing it through a flame two to three times with the smeared slide away from the flame. If desired, this dried and fixed amount may be stained and the preparation dried again for observation under the microscope.

Micrometry: Measurement of the Size of Objects by Compound Microscope

The size of objects viewed under the compound microscope can be accurately determined using a micrometer. The latter consists of two scales, the eyepiece scale, (also called 'graticule' or 'ocular') and the stage micrometer scale. The eyepiece scale is calibrated with the help of stage micrometer and the former is then used for measurements.

The eyepiece scale is placed inside the microscope eyepiece, and the stage micrometer on the microscope stage. The scale on the latter is exactly 1 mm long and divided into 100 divisions, so that each division is 10 μm. As stated earlier, the stage micrometer is used to calibrate the eyepiece scale.

(i) Calibration

1. It is noted first that which objective lens is in use on the microscope.

2. Stage micrometer is positioned in such a way that it is in the field of view.

3. The eyepiece is rotated so that the two scales, the eyepiece or ocular scale and the stage micrometer scale, are parallel.

4. The stage micrometer is now moved so that the first division marks of the two scales are in line. One can now see how many divisions on the eyepiece scale as well as on the stage micrometer scale correspond to each other. Since 1 division on the stage micrometer equals 10 μm, one can find the value of one division of the eyepiece scale.For instance, in illustration 'iii' of four divisions on the eyepiece scale equal 10 divisions (i.e., 100 μm) of the stage micrometer scale; 1 division on the eyepiece scale = 25 mm for the particular objective lens used in this case.

Diagrammetic representation of micrometry

Above positions are repeated using objective lenses and following informations are recorded on an adhesive label. Information recorded on adhesive label is stuck to the base of the microscope for future reference: