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The behavior of fluorescence anisotropy and polarization in systems with multiple dyes is well known. Homo-FRET and its consequent energy migration cause the fluorescence anisotropy to decrease as the number of like fluorophores within energy transfer distance increases. This behavior is well understood when all subunits within a cluster are saturated with fluorophores. However, incomplete labeling...
Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive...
We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and...
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