Quantitative scheme for full-field polarization rotating fluorescence microscopy (PROM) using a liquid crystal variable retarder

Published

May 14, 2015

Author(s)

John F. Lesoine, Ji Y. Lee, Hyeong G. Kang, Matthew L. Clarke, Robert C. Chang, Ralph Nossal, Jeeseong C. Hwang

Abstract

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.

Citation

Review of Scientific Instruments

Pub Type

Journals

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Keywords

polarization microscopy, fluorescence imaging, liquid crystal variable retarder, fluorescent analog, DiI, absorption dipole, lipid domain, membrane dynamics, cellular process, polarization rotating microscopy, PROM

Citation

Lesoine, J. , Lee, J. , Kang, H. , Clarke, M. , Chang, R. , Nossal, R. and Hwang, J. (2015), 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 October 7, 2025)