What factors affect the quality of image obtained using a light microscope? How - Answers highly appreciated... (Jan/06/2009 )

What factors affect the quality of image obtained using a light microscope? How can staining techniques be used to enhance the quality of the image?

Asked in exam so really want to know the answer from professionals like people in this forum. Answers highly appreciated. Thanks.

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It would help if you were to copy the precise question here. I feel it is a vague question to begin with. All steps prior to microscopy you do have to be optimized to get the best image.

0. Proper section thickness (ideally between 2 and 7 micron thickness).
1. Optimized fixation
2. Optimized staining
3. Use of counter-stains (for example, if you are looking at cytoplasm, also counter-stain nuclei)
4. Proper mounting (no bubbles, clean coverslips etc).
5. Proper storage.

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do you think in terms of cells or tissues? quality of preparation of cells/tissues must be excellent; less specific stanings with chromophores are possible but often obsolet; immunostainings for tissues are possible to inspect with Light Microscope

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Thanks. I was referring to cells actually.

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Obvious ones are the thickness of a specimen and the mounting medium used. Types of lighting are important too, bright field, dark field, fluorescence. Is koehler illumination properly set up for standard bright field illumination. The colour temperature of the the bulb is important too if you want true white light.

If you are talking about pictures then: the quality of the film, the ISO rating and the exposure time are all important. Now a days with digital cameras the type of sensor and the sensor size are important, ideally you would use a high megapixel sensor with a large area (anything over about 2.5 cm is getting to be OK) and one that is CCD and cooled helps too.

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