Real-Time Fluorescence Polarization Microscopy For Probing Local Distributions Of Biomolecules

We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY)-labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.