Bright-Field Microscope

In actual, practice, the ‘ordinary microscope’ is usually refereed to as a bright-field micro-

scope by virtue of the fact that it gives rise to a distinct dark image against a brighter background. Description : Bright-field microscope essentially comprises of a strong metalic body with a

base and an arm to which the various other components are duly attached as shown in Fig. 4.3 (a). It is provided with a ‘light source’ either an electric bulb (illuminator) or a plano-concave mirror strategi-

cally positioned at the base. Focusing is accomplished by two knobs, first, coarse adjustment knob, and secondly, fine adjustment knob which are duly located upon the arm in such a manner that it may move either the nosepiece or the stage so as to focus the image sharply.

In fact, the upper segment of the microscope rightly holds the body assembly to which is attached a nosepiece or eyepiece(s) or oculars. However, the relatively advanced microscopes do possess eyepieces meant for both the eyes, and are legitimately termed as binocular microscopes. Importantly, the body assembly comprises of a series of mirrors and prisms in order that the tubular structure very much holding the eyepiece could be tilted to afford viewing convenience. As many as 3 to 5 objectives having lenses of varying magnifying power that may be carefully rotated to such a position which helps in clear viewing of any objective help under the body assembly. In the right ideal perspective a microscope must be parfocal*.

In order to achieve high magnification (× 100) with markedly superb resolution, the lens should be of smaller size. Though it is very desired that the light travelling via the specific specimen as well as the medium to undergo refraction in a different manner, at the same time it is also preferred not to have any loss of light rays after they have gained passage via the stained specimen. Therefore, to preserve and maintain the direction of light rays at the maximum magnification, an immersion oil is duly placed just between the ‘glass slide’ and the ‘oil immersion objective lens’, as depicted in Fig. 4.3 (b). Interestingly, both ‘glass’ and ‘immersion oil’ do possess the same refractive index ; and, therefore, rendering the ‘oil’ as an integral part of the optics of the glass of the microscope. In fact, the ‘oil’ exerts more or less the identical effect as would have been accomplished by enhancing the diameter of the ‘objective’ ; and, therefore, it critically and significantly elevates the resolving power of the lenses. Thus, the condenser gives rise to a bright-field illumination.

Line of vision Ocular lens Path of light Prism Body tube Objective lenses Specimen Condenser lenses Illuminator Base with source of

illumination Light source

Iris diaphragm Condenser lenses Condenser Immersion oil Oil immersion objective lens Unrefracted light Refracted light without immersion oil

(a)

(b) The path of light (bottom to top)

Air

Glass slide

Fig. 4.3. (a) Diagramatic sketch of the Path of light in a Bright-Field Microscope.

Fig. 4.3. (b) Refraction in a Bright-Field Microscope Employing an Oil Immersion Objective Lens. [Adapted From : Tortora GJ et. al. Microbiology an Introduction, The Benjamin/Cummings Publishing Co., Inc., New York, 5th edn, 1995].

Phase plate

Bacterium Ray deviated by

specimen is 1/4 wavelength out of phase. Deviated ray is 1/2 Wavelength out of phase. Deviated and undeviated rays cancel each other out.

Fig. 4.6. Production of Contrast in Phase Microscopy Comparison of Contrasting Light Pathways of Bright-field, Dark-field, and Phase-contrast Microscopes.

The ensuing contrast-light pathways of bright-field, dark-field, and phase-contrast microscopes have been explicitely illustrated in the following Fig. 4.7.

Ocular Lens Objective Lens Specimen Condenser Lens Light Eye Eye Ocular Lens Objective Lens Specimen Condenser Lens Light

Only light reflected by the specimen is captured by the objective lens Unreflected Light Opaque Disc Ocular Lens Objective Lens Specimen Condenser Lens Annular Diaphragm

Diffracted Light (phase altered by specimen) Undiffracted Light (phase unaltered by specimen)

Diffraction (phase) Plate Eye

Light

(a) (b) (c)

Fig. 4.7. Comparison of Light Pathways of Bright-field, Dark-field, and Phase-contrast Microscopes [(a), (b) and (c)].

(a) Bright-field : Shows the path of light in the bright-field microscopy i.e., the specific kind of illumi- nation produced by regular compound light microscopes.

(b) Dark-field : Depicts the path of light in the dark-field microscopy i.e., it makes use of a special condenser having an opaque disc which categorically discards all light rays in the very centre of the beam. Thus, the only light which ultimately reaches the specimen is always at an angle ; and thereby the only light rays duly reflected by the specimen (viz., gold rays) finally reaches the objective lens.

(c) Phase-contrast : Illustrates the path of light in the phase-contrast microscopy i.e., the light rays are mostly difracted altogether in a different manner ; and, therefore, do travel various pathways to reach the eye of the viewer. Thus, the diffracted light rays are duly indicated in gold ; whereas, the undiffracted light rays are duly shown in red.