Light microscope
Microscope is the instrument most characteristics of the microbiological laboratory. The magnification it provides enables us to see microorganisms and their structures otherwise invisible to the naked eye. The magnification attainable by microscope ranges from X100 to X400, 000. Several different kinds of microscopy are available many techniques have been developed by which specimens can be prepared for examination. Each type of microscopy and each method of preparing specimen offers advantages for demonstration of specific morphological features.
There are two fundamentally different types of microscope. Light microscope and electron microscope. Light microscope uses a series of glass lenses to focus light in order to form an image. Maximum magnification attained is X1, 500. Electron microscope uses electromagnetic lenses to focus a beam of electrons. Maximum magnification attained is X400, 000.
Both living and dead specimens can be viewed with light microscope and often in real color.
Only dead microorganisms are viewed with electron microscope and never in real color.
Magnification is not the best measure of a microscope. Resolution or resolving power, the ability to distinguish two adjacent points as distinct and separate in a specimen, is much more reliable estimate of a microscope’s utility. Greater magnification without greater resolution i.e., mere increase in size without the ability to distinguish structural details is not beneficial. The largest magnification produced by a microscope may not be the most useful because the image obtained may be unclear or fuzzy. Magnification beyond the resolving power is of no value and is called empty or useless magnification. The resolving power of a microscope is a function of the wavelength of light used and the numerical aperture of the lens system. Total magnification of the system is determined by multiplying the magnifying power of the objective by that of eyepiece.
Resolution of light microscope is 0.5 µm approximately.
Resolution of electron microscope is upto 1 nm
In bright field microscopy, the microscopic field is brightly lighted and microorganisms appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light but staining them with dye greatly increases their light absorbing ability resulting in greater contrast and color differentiation.
All modern light microscopes are made up of more than one glass lens in combination. The major components are condenser lens, the objective lens and the eyepiece lens. Each of these components is in turn made up of combinations of lenses which are necessary to produce magnified images.
There are two basic types of compound light microscope stand- upright and inverted microscope. Upright microscope is for viewing specimens. Condenser lens and light source are below specimen. Stage is movable.
Inverted microscope is for manipulation of specimen directly on stage. E.g,. microinjection of macromolecules into tissue culture cells, invitro fertilization of eggs etc. condenser lens and source of light are above specimen. Objective is movable.
Numerical aperture is a measure of the ability of lens to collect light from the specimen. Lenses with low numerical aperture collect less light than those with higher numerical aperture. Higher numerical aperture objective yields best resolution. The angle theta subtended by optical axis and the outermost rays still covered by objective lens is the measure of aperture of objective. It is half aperture angle.
The magnitude of this angle is expressed as a sine value.NA=n X sin theta
Sin theta=sine value of half aperture angle
N=refractive index of the medium filling the space between front lens and coverslip.
For dry objective n=1=refractive index of air
For oil immersion n=1.56=refractive index of oil immersion which is equal to refractive index of glass.
The wavelength of light used in optical microscope is also limited. The shorter the wavelength of illuminating light, the higher is the resolving power of the microscope. Visible wave length of light rages between 400 nm and 750 nm. By using ultraviolet light as light source, the resolution can be improved.
Greatest resolution using visible light=200 nm
Greatest resolution using ultraviolet light=100 nm
Greatest resolution in light microscopy is obtained using shortest wavelength of visible light and objective with maximum numerical aperture.
Dark field microscopy
It produces image of brightly illuminated objects on a black background. Brilliant illumination of objects is accomplished by equipping light microscope with a special kind of condenser that transports a hollow cone of light from the source of illumination. Most of the light directed through the condenser does not enter the object. Therefore, field is dark. However, some of the light rays will be diffracted (scattered) if transparent medium contains objects such as microbial cells. This diffracted light will enter the objective and reach the eye. Thus objects or microbial cells appear bright. It is used for viewing motility and outlines of objects in liquid media such as living spermatozoa, microorganisms, cells growing in tissue culture.
Spirochaetes Treponema pallidum causative agent of syphilis STD can’t be seen in Gram stained smears, Borrelia, Leptospira, Vibrio.
Fluorescence microscopy
In fluorescence microscopy, ultraviolet light which has very short wavelength and is not visible to the eye, is used to illuminate organisms, cells, particles which have been previously stained with fluorescing dyes called fluorochromes. These dyes are able to transform the invisible short wavelength ultraviolet light into longer wavelength visible light. The fluorescent stained particles appear glowing against a dark background.
Two types of fluorescence microscopy are used in medical laboratory work- transmitted light fluorescence (TLF) and incident light fluorescence (ILF) also called epifluorescence.
Transmitted light fluorescence –Short wavelength light from a fluorescence lamp such as mercury vapor or quartz halogen lamp passes through primary or excitation filter which removes all unwanted color or wavelength of light and passes only those that are required. The transmission of this filter must match the emission peak of the fluorochromes being used. The light is then brought to the specimen by a dark field immersion condenser. The fluorescence which is given off by the specimen passes through a secondary or barrier filter located between the objective and the eye which filters of all the light other than fluorescence wavelength specific to the specimen
Incident light fluorescence
This involves illuminating the specimen fro above (epifluorescence) using a dichroic mirror which reflects selectively the shorter wavelength radiation and transmits the longer wavelength fluorescence i.e., it is transparent to wavelength above given value and opaque to those below.
The light passes through an excitation filter and is directed onto dichroic mirror located above specimen. The mirror reflects short wavelength excitation light on specimen. The visible light from the reflected specimen passes back through the objective to the dichroic mirror which transmits the longer fluorescent wavelength. A barrier filter ensures that only the fluorescence wavelength specific for the specimen reach the eyepiece. No condenser needed for incident light fluorescence.
Phase contrast microscope
It is extremely valuable for studying living unstained cells. It uses a conventional light microscope fitted with phase contrast objective and phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell which differ only slightly in their refractive indices or thickness.
As light passes through a medium other than vacuum, interaction with this medium causes its phase to change in a way which depends on properties of the medium. These changes in phase carry large amount of valuable information. However, these changes in phase are not easily observed by human eye. Therefore, optical mechanism is employed to translate variation in phase into corresponding change in brightness of structures and hence is detectable by eye.
Illumination produced by tungsten halogen lamp is focused on a specialized condenser annulus positioned below sub stage condenser. Light passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures present in specimen. Undeviated and diffracted light collected by the objective is segregated by phase plate and focused at the intermediate image to form final phase contrast image.
