This page introduces microscope methods for observation and analysis in cell biology.
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There are 3 types of light microscopes used in the lab. The theoretical limit of resolution of the light microscope is approximately 0.2 microns.
Bright Field microscope for looking at fixed and stained tissue sections or cell cultures.
Dissecting microscope for fine dissection of tissues prior to analysis or cell culture. These are stereo-microscopes that give a three-dimensional view of a specimen. They have a limited magnification (100-150 times). These microscopes are often used in conjunction with a cold fiber optic light source, to prevent heating/heat damage of the specimen tissue.
A cold fiber optic light as the name suggests, directs a light source over a distance through fiber optics, thereby separating the hot light from the specimen.
See also commercial resources: Meiji EMZTR Zoom Stereo Microscope.
Phase Contrast inverted microscope for looking at living cells in cell culture. These microscopes shift light waves "out of phase" from each other, then this change is converted into amplitude changes (brightness) of the subject. This is seen as a bright refractive "halo" around living cells, which is abscent from dead or damaged cells.
See also CBL Methods: Microscope Calibration
See also commercial resources: Olympus IX71 and CKX microscopes.
Fluorescent microscopes can be used with either fixed or living cells. There are many different fluorescent indicators, labels and even autofluorescence that can be used in both fixed and living cells.
An excellent introduction can be found in the Molecular Probes handbook- Introduction to Fluorescence Techniques
Living (and fixed) cells may also "autofluoresce" due to the presence of some biological compounds within the cell or media (tryptophan, NADH, NADPH, riboflavins, flavin coenzymes, lipofuscin, iminopropenes, phenol red, etc). Some fixation procedures will also increase the level of autofluorescence.
Some research also suggests that normal, pre-neoplastic and neoplastic cells/tissues differ in the levels orf autofluoresence.
Also detection of fluorescent antibodies (fluorescein-conjugated) signal in 515-555 nm range "green" fluorescence) can include both fluorescein emission and part of cellular autofluorescence. Some researchers have applied a subtraction of the 580 nm "red" autofluorescence to get a fluorescein only signal.
See also CBL Methods: Immunochemistry
1. Aubin, JE. Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem 27:36-43, 1979. uids=220325
2. Benson RC, Meyer RA, Zaruba ME, McKhann GM. Cellular autofluorescence: Is it due to flavins? J Histochem Cytochem 27:44-48, 1979. uids=438504
3. Alberti S, Parks DR, Herzenberg LA. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry. 1987 Mar;8(2):114-9. uids=3556100
A confocal microscope has the capablity of isolating and collecting a plane of focus from within a cell or tissue, eliminating the out of focus "haze" normally seen on a fluorescent microscope.
Electron microscopes use a focused beam of electrons rather than light to "illuminate" a specimen. As such, images are generally of dead cells, that have been fixed and contrasted with osmium tetroxide (OsO4).
The 2 main types are:
Transmission Electron Microscope (TEM) see through the specimen (developed by Max Knoll and Ernst Ruska in Germany in 1931).
Scanning Electron Microscope (SEM) involve "scanning" a beam of electrons across the sample.
Atomic Force Microscopy (AFM) and Scanning Tunneling Electron Microscope are 2 new developing technologies.
Scanning Electron Microscope (SEM)
Transmission Electron Microscope (SEM)
Search all NCBI Books for the term "microscope"(520 results)
Search MBoC4 for the term "microscope" (120 results)
The Cell Tools of Cell Biology - light microscope
These commercial links are for educational purposes only and do not reflect endorsement of a specific product.
Olympus Microscopy Resource Center
Olympus IX71 Inverted Phase Microscope Information
Olympus IX71 Inverted Phase Microscope Brochure
Laser Scanning Confocal Microscope Simulator
Fluorescence Excitation and Emission Fundamentals
This page introduces the use of microscopes in the Lab and links to useful external microscope resources. While not all of the above microscopic techniques may be used routinely in the Lab, a broad understanding of the use and theoretical background of each technique is essential.
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