PubMed · 8574709
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1995. Biophysical methods.. https://doi.org/10.1016/0959-440x(95)80067-0
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The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.
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Molecular interactions occurring on or near cell membrane surfaces are expected to have different properties from those occurring in bulk solutions. One particularly useful technique for studying surface-associated processes at the molecular level is total internal reflection fluorescence. In this method, the evanescent field from an internally reflected excitation source selectively excites fluorescent molecules on or near a surface. Evanescent excitation has been used recently with a variety of techniques in fluorescence microscopy and spectroscopy to probe the fundamental physicochemical properties of biochemical reactions at natural or model biological surfaces. These studies are providing enhanced understanding of cellular function. Several recent developments in total internal reflection fluorescence methodology from other fields are likely to find future application in cellular biophysics.