Definition of Microscopy:
According to Wikipedia "Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye."
*Microscopy is an important component of diagnostic microbiology.*
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| Microscopic Analysis of some organelles |
Microscopic methods:
1. Light microscopy
2. Electron microscopy
3. Scanning probe microscopy
1. Light microscopy
**A light microscope
refers to the used of any kinds of microscope that uses natural or
artificial transmitted light as the source of light to observe specimens.
In light microscopy, light typically passes through a specimen and then through a series of magnifying lenses. Here microscopy resolving power is an important component.**
Different types of Light Microscopy include
· Bright-field Microscopy
· Dark-field Microscopy
· Dark-ground Microscopy
· Phase-contrast Microscopy
· Interference Microscopy
· Fluorescence Microscopy
[Microscope is an optical instrument used to magnify minute objects or micro-organism which cannot be seen by naked eye.
In general, Microscope is used in microbiology for two basic purposes:
· The initial detection of microbes and
· The preliminary or definitive identification of microbes.]
A. Bright-field Microscopy:
Bright field Microscopy always refers to as ordinary Light Microscope is the most common form of Light Microscopy that uses a compound Light Microscope.
A compound
light microscope primarily consists of a compound lens system
that contains a number of objective lens.
With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium.
Many bacteria are difficult to see well because of their lack of
contrast with the surrounding medium
It may be used
to examine either wel-films or “hanging drop” for demonstration
of the motility of the flagellated bacteria and protozoa. It is also useful for
demonstration of the structural details.
B. Dark-ground (Dark-field) Microscopy:
The Dark ground microscopy uses of Dark Ground Microscope, a special type of compound Light Microscope.
This Microscope uses reflected light instead of transmitted light used in the ordinary Light Microscope.
- It prevents light from falling directly on the objective lens with the result that the micro-organisms appear brightly stained against a dark background.
- It is useful for very thin bacteria (such as spirochetes) not visible under ordinary illumination, since the reflection of Light Microscope makes them appear larges.
- It is also helpful for demonstration of motility of flagellated bacteria and protozoa.
This technique of Microscopy is particularly valuable for
observing organisms such as Treponema pallidum, a spirochete which is
less than 0.2 mm in diameter and therefore cannot be observed with direct
light.
C. Phase-contrast Microscopy:
The Phase-contrast Microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them.
Phase-contrast Microscopy is based on the wave nature of light rays, and the fact that the light rays can “in phase” (their peaks and valleys match) or “out of the phase”.
Phase-contrast
Microscopy improves the contrast;
the internal structures of a cell become more sharply defined and make
evident the structure within cells that differ in thickness or refractive
index. It is used for studying unstained cells, detailed examination
of internal structures in living micro-organisms.
*It is used to study Amoeba and Trichomons.
D. Fluorescent microscopy:
Fluorescent Microscopy is based on the principle that the specimens stained with “fluorescent dye” when exposed to ultraviolet light result in the emission of longer wavelength of light.
Fluorescence needs a “Fluorescence Microscope”
fitted with an ultraviolet source.
It is used for “direct demonstration” of the antigen of a pathogen in clinical specimens by direct “fluorescent antibody test” and also used for estimation of antibodies in the serum by a direct fluorescent antibody test.
Fluorescence Microscopy is widely used in clinical diagnostic microbiology. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis.
When the dye is applied to a specimen suspected of containing M tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background.
E. Interference Microscopy/Confocal Microscopy:
This is another specialized application of Light Microscopy used for demonstrating cell organelles.
In Confocal Scanning Laser Microscopy, a laser beam is bounced off a mirror that directs the beam through a scanning device.
Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a given vertical layer within the specimen.
By precisely
illuminating only
a single plane of the specimen, illumination intensity drops off rapidly above and below the plane of focus, and stray light
from other planes of focus are minimized.
[Thus, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the laser beam.]*
Its main use :
1. To obtain three
dimensional images of entire cell; by monitoring the
distributions and concentrations of substances such as ATP and calcium ions.
2. To evaluate cellular
physiology.
Thus, images obtained from different layers can be stored and then digitally overlaid to reconstruct a three-dimensional image of the entire specimen.
2. Electron Microscopy
The high resolving power of Electron Microscopes has enabled scientists to observe the detailed structures of prokaryotic and eukaryotic cells.
Electron microscopy utilizes the beam of electrons instead of a beam of light used in the Light Microscope.
Rather than using glass lenses, visible light and the eye to
observe the specimen, the Electron Microscope uses *electromagnetic
lenses*, and a fluorescent screen to produce the magnified image.
There are mainly two types of Electron Microscope in general use:
1.
Transmission
Electron Microscope (TEM):
In Transmission Electron Microscope, electrons
like light pass directly through the specimen that has been prepared by thin sectioning
freeze fracturing or freeze etching.
*It is used to observe fine details of cell structure.*
2.
Scanning Electron
Microscope:
The Scanning Electron Microscope scans a beam of
electrons back and forth over the surface of the specimen; producing three
dimensional views of the surface of whole micro-organisms.
3. Scanning Probe Microscopy
Another Electron
Microscopy is Scanning Probe Microscopy. It is also very useful and
popular instrument, in the latest modern research projects and thesis.
It uses the measure surface features by moving a sharp probe
over the object’s surface.
Scanning probe Microscopes maps the bumps and the valleys of a surface on an atomic scale.
Their resolving
power is much greater than the Electron Microscope and the samples do not
need special preparation as they do for Electron Microscopy.
Among the new scanned probe Microscopes are:
1. Scanning Tunneling Microscopy (STM): They are used to provide indirectly detailed views of molecules such as DNA.
2. Atomic Force Microscope (AFM): They produce three-dimensional images of the surface of a molecule.
*Interactions between proteins of the bacterium, Escherichia coli can be studied with the atomic force microscope.
Mainly Electron microscopy is difficult for its operating system and the maintenance cost is also a great issue. Operators must require special training. Maybe it can not be used for personal use in some countries.
But Light microscopic instruments does not require any type of special requirements and it can be used for personal or semi-commercial use.
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