Microspectrophotometry: The Technique and Some of Its Pitfalls
Caspersson, by combining the procedures of microscopy and spectrophotometry, founded the technique of microspectro-photometry (MSP) nearly forty years ago. Whereas the light microscope affords the visual identification of subcellular elements, spectroscopy, by virtue of the fact that many substances possess characteristic absorption spectra, allows the determination of the presence and concentration of known substances by the measure of light fluxes transmitted at various wavelengths through structures of known thickness. Thus, he reasoned, the absorption of light by cellular components may be used as a tool for their qualitative and quantitative chemical analyses. Caspersson (1940, 1950) succeeded not only in designing the first microspectrophotometer and setting down the principles of the method but also in identifying nucleic acids (λmax ≃ 260 nm) in the nucleus and proteins with cyclic amino acids (λmax ≃ 280 nm) in the cytoplasm of isolated cells.
KeywordsNumerical Aperture Outer Segment Visual Pigment Linear Dichroism Characteristic Absorption Spectrum
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