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Biophys J, April 2000, p. 1703-1713, Vol. 78, No. 4
*EVOTEC BioSystems AG, D-22525 Hamburg, Germany, and
Institute of Experimental Biology, Harku 76902, Estonia
A method of sample analysis is presented which is based
on fitting a joint distribution of photon count numbers. In
experiments, fluorescence from a microscopic volume containing a
fluctuating number of molecules is monitored by two detectors, using a
confocal microscope. The two detectors may have different
polarizational or spectral responses. Concentrations of fluorescent
species together with two specific brightness values per species are
determined. The two-dimensional fluorescence intensity distribution
analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA,
binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an
example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in
stained vesicles has been studied. The presented method is unusually
accurate among fluorescence fluctuation methods. It is well suited for
monitoring a variety of molecular interactions, including receptors and
ligands or antibodies and antigens.
Biophys J, April 2000, p. 1703-1713, Vol. 78, No. 4
© 2000 by the Biophysical Society 0006-3495/00/04/1703/11 $2.00
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