Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in live cells

Molly McQuilken¹, Shalin B. Mehta², Amitabh Verma², Grant Harris², Rudolf Oldenbourg², and Amy S. Gladfelter¹

¹Department of Biological Sciences, Dartmouth College, Hanover, NH 03755
²Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA 02543

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 non-destructive 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 (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.

Keywords: polarized fluorescence, cytoskeleton, image analysis, OpenPolScope

Current Protocols in Cell Biology
67:4.29.1-4.29.13
(2015)
•doi: 10.1002/0471143030.cb0429s67

Downloads specific to this article:

Micro-Manager 1.4.15 (September 23, 2013)

Micro-Manager is a software package for control of automated microscopes. Micro-Manager is bundled with the image processing application ImageJ and provides a comprehensive, freely available, imaging solution.

Windows 32-bit, 50 MB
Windows 64-bit, 50 MB
Mac OS X (Universal Binary), 46 MB

OpenPolScope 2.0 (December 03, 2014)

OpenPolScope software is a plugin for Micro-Manager and implements instrument control, image acquisition and processing algorithms for rapidly imaging optical anisotropies in three different modes: birefringence, polarized fluorescence, and diattenuation.

Windows 32/64 bit, 50.3 MB
Mac OS X (Universal Binary), 45.5 MB