Open AccessPolarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells
Author(s) -
McQuilken Molly,
Mehta Shalin B.,
Verma Amitabh,
Harris Grant,
Oldenbourg Rudolf,
Gladfelter Amy S.
Publication year - 2015
Publication title -
current protocols in cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.149
H-Index - 38
eISSN - 1934-2616
pISSN - 1934-2500
DOI - 10.1002/0471143030.cb0429s67
Subject(s) - microscopy , fluorescence microscope , polarizer , fluorescence , cytoskeleton , software , fluorescence lifetime imaging microscopy , microscope , nanotechnology , optics , materials science , computer science , chemistry , physics , birefringence , cell , biochemistry , programming language
The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open-access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze polarized fluorescence.