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Proceedings Paper

Real-time fluorescence polarization microscopy for probing local distributions of biomolecules
Author(s): Ji Youn Lee; John F. Lesoine; Jeffrey R. Krogmeier; Hyeonggon Kang; Matthew Clarke; Robert Chang; Dan L. Sackett; Ralph Nossal; Jeeseong Hwang
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Paper Abstract

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.

Paper Details

Date Published: 28 February 2011
PDF: 6 pages
Proc. SPIE 7891, Design and Quality for Biomedical Technologies IV, 78910Z (28 February 2011); doi: 10.1117/12.875612
Show Author Affiliations
Ji Youn Lee, National Institute of Standards and Technology (United States)
National Institutes of Health (United States)
John F. Lesoine, National Institutes of Health (United States)
Jeffrey R. Krogmeier, National Institute of Standards and Technology (United States)
Life Technologies, Inc. (United States)
Hyeonggon Kang, National Institute of Standards and Technology (United States)
Matthew Clarke, National Institute of Standards and Technology (United States)
Robert Chang, National Institute of Standards and Technology (United States)
Dan L. Sackett, National Institutes of Health (United States)
Ralph Nossal, National Institutes of Health (United States)
Jeeseong Hwang, National Institute of Standards and Technology (United States)


Published in SPIE Proceedings Vol. 7891:
Design and Quality for Biomedical Technologies IV
Ramesh Raghavachari; Rongguang Liang, Editor(s)

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