Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy

Albert H. Gough; D. Lansing Taylor

doi:10.1117/12.182735

17 August 1994 Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy

Albert H. Gough, D. Lansing Taylor

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182735
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

Fluorescence polarization anisotropy can be used to determine the rotational mobility of a fluorescent analog, detect anisotropic orientation distributions, or measure the fluorescence lifetime of a fluorophore. Steady state fluorescence anisotropy can be simply measured in a standard fluorescence microscope equipped with excitation and emission polarizers and therefore, two dimensional maps of fluorescence anisotropy can be easily acquired. We are using steady state fluorescence anisotropy imaging microscopy to study the biochemistry of cell motility. The optimum fluorophore for fluorescence polarization measurements has a fluorescence lifetime that is comparable to the rotational correlation time of the molecule of interest. In order to make imaging measurements with high sensitivity and reasonable time resolution, however, this general rule has to be adjusted, and we have found that FITC-calmodulin has a useful combination of the above features. Calmodulin is a key regulatory protein, that is proposed to be involved in the regulation of the actin-myosin II based force generation in non-muscle cells. The rotational mobility of macromolecules is very sensitive to molecular interactions, yet is relatively insensitive to any surrounding gel matrix. We have taken advantage of this feature to map FITC-calmodulin interactions in the complex cytomatrix in living cells by steady state Fluorescence Anisotropy Imaging Microscopy (FAIM). In addition, we are investigating the use of FAIM for mapping variations in molecular orientation distributions, and fluorescence lifetime distributions.

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Albert H. Gough and D. Lansing Taylor "Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182735

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KEYWORDS

Luminescence

Analog electronics

Molecules

Proteins

Anisotropy

Fluorescence anisotropy

Molecular interactions

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