Quantitative scheme for full-field polarization rotating fluorescence microscopy (PROM) using a liquid crystal variable retarder
John F. Lesoine, Ji Y. Lee, Hyeong G. Kang, Matthew L. Clarke, Robert C. Chang, Ralph Nossal, Jeeseong C. Hwang
We introduce real-time, full-field, polarization rotating fluorescence microscopy (PROM) to monitor the absorption dipole orientations of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate linearly polarized light prior to its being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images is obtained from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. The fluorescent lipid-like analog used in this study is a molecule that is labeled by a tetramethylindocarbocyanine (DiI) fluorophore in its head group. We adapt a phase-shifting interferometry algorithm to PROM measurements to determine the orientation of the absorption dipole of DiI molecules and the fluorescence intensity and normalized amplitude of the polarized fluorescence signal. A contrast in the normalized amplitude is observed on the cell membrane but the amplitude of the labeled nucleus is zero, which indicates randomized orientations over the length scale of a single pixel. This instrument provides a low cost calibrated method that can be coupled to existing fluorescence microscopes to perform investigations of characteristic cellular processes.
, Lee, J.
, Kang, H.
, Clarke, M.
, Chang, R.
, Nossal, R.
and Hwang, J.
Quantitative scheme for full-field polarization rotating fluorescence microscopy (PROM) using a liquid crystal variable retarder, Review of Scientific Instruments, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=908548
(Accessed December 9, 2023)