What Are The Different Types Of Transmission Microscopy?
With the increase in clinical research, more and more input has been done to improve the visualisation skills to view cells. This has lead to many variations to bright field of transmission microscopy. Hence many modifications of light microscopy that have specialized applications have been developed.
Dark Field Microscopy:
In dark-field microscopy, the specimen under observation is illuminated from the side and only scattered light is allowed to enter the objective lens which appears in bright objects aligned with a dark background. This is done through the utilization of an annular aperture that produces a hollow cone of light which does not enter the objective lens. Some of the light that hits the objects within the specimen is diffracted into the objective lens. The images so produced by dark-field microscopy have low resolution and most details cannot be seen clearly. Dark-field microscopy is especially valuable for visualization of small particles such as bacteria.
Phase-Contrast Microscopy:
Phase-contrast microscopy allows the objects that differ slightly in refractive index or thickness to be distinguished between unstained or living cells. Distinction in the thickness or refractive index within the specimen marks in a differential retardation of light which shifts the phase or deviates the direction of the light. During phase-contrast microscopy, the phase disparity is converted to intensity differences by special objectives and condensers.
Fluorescence Microscopy
In fluorescence microscopy a fluorochrome is energized with ultraviolet (UV) light and the resulting observable fluorescence is viewed. This creates a bright image in a dark background.
Confocal microscopy
This utilises the objective lens as both the objective and the condenser. This permits the illuminating light to be focused on a relatively thin plane. In addition, a ‘pin-hole’ is utilised to further reduce the light coming from other planes. Minimizing the intrusion from other planes boosts apparent resolution.
Preparation of a sample:
The specimen can be viewed by placing it on a glass microscope slide covered under a glass cover slip. However, fixation is a process by which cells are preserved and stabilized on the glass slide. A few common fixatives include acids, formaldehyde, organic solvents, and glutaraldehyde. These treatments fix the macromolecules in position. Thick samples, such as tissue specimens and biopsies, need to cut into thin sections. After following the fixation stage, the sample or cells are embedded into a supporting medium like paraffin which is a common embedding medium for light microscopy as well as various plastic resins. Sectioning is carried out with a microtome that cuts the specimen into thin sections of a specified thickness.
Staining:
The images that are produced by microscopy depend upon different components in the sample that are interacting with and obstructing the light waves differentially. Biological samples are fairly homogeneous (i.e., carbon-based polymers) and do not greatly hold up light. Therefore, it is often necessary to stain cells with dyes to offer more contrast. The stained sub-cellular components will differentially soak up the light waves and result in less light reaching the eye, and thus appear darker.
