title: Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire.
authors.forename: Molly
authors.initials: M
authors.lastname: McQuilken
authors.affiliation: Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts.
authors.forename: Shalin B
authors.initials: SB
authors.lastname: Mehta
authors.affiliation: Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts.
authors.forename: Amitabh
authors.initials: A
authors.lastname: Verma
authors.affiliation: Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts.
authors.forename: Grant
authors.initials: G
authors.lastname: Harris
authors.affiliation: Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Oldenbourg
authors.affiliation: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Gladfelter
mesh_terms: D003599:Cytoskeleton / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D050296:Microbial Viability; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D058112:Septins / Q000378:metabolism; D012984:Software
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D058112:Septins
keywords: OpenPolScope; cytoskeleton; image analysis; polarized fluorescence