Light microscopy

Light microscopy:

The compound microscope that is most generally employed today has some lenses which magnify the image of a specimen. The total magnification of the object is a product of the magnification of the separate lenses; when the objective lens magnifies 100 -fold and the eye piece magnifies 10- fold, then the final magnification recorded by the human eye or on film will be 1000- fold.

The limit of resolution of a light microscope employing visible light is around 0.2 µm (i.e., 200nm). No matter how many times the image is magnified, the microscope can never determine objects which are less than ≈ 0.2 µm separately or reveal details smaller than ≈  0.2 µm in size.

The sample for light microscopy are generally fixed, sectioned and stained. Specimens for light microscopy are generally fixed with a solution merging alcohol or formaldehyde, compounds which denature most protein and nucleic acids. Generally the sample is then embedded in plastic or paraffin and cut into thin sections of one of a few micrometers thick employing a microtome. Then such sections are stained employing suitable stains.

 

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