Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells

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

1 Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, 2 Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.29
DOI:  10.1002/0471143030.cb0429s67
Online Posting Date:  June, 2015
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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. © 2015 by John Wiley & Sons, Inc.

Keywords: polarized fluorescence; cytoskeleton; image analysis; OpenPolScope

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Medium with low fluorescence for imaging
  • Agarose
  • Overnight culture of Saccharomyces cerevisiae
  • Septin‐GFP expressing yeast can serve as a control for instrument set‐up as the signal is robust and reproducible (DeMay et al., , b)
  • VALAP (see recipe)
  • Liquid crystal (LC) based universal polarizer with mounting adaptor and electronic controller with operating wavelength of 450 to 700 nm (hardware can be purchased from OpenPolScope Resource)
  • Alternative to LC: five linear polarized filters (Chroma 21033a) in filter wheel (Ludl Electronic Products, cat. no. 99A075) and UV blocker 420 nm LP emission filter 32 nm (Chroma, cat. no. E420LPv2‐32)
  • Wide‐field microscope (for best results, use strain‐free oil immersion objective lenses and condenser optics; any brand)Slide with sheet polarizer with known transmission orientation, preferably parallel to an edge
  • OpenPolScope software (full installation, http://www.openpolscope.org)
  • Microwave
  • 1.5‐ml microcentrifuge tubes
  • Heat block
  • Depression slides
  • Coverslips for mounting S. cerevisiae sample
  • Micro‐Manager (http://www.micro‐manager.org) (Edelstein et al., )
  • Digital CCD camera supported by Micro‐Manager software
  • ImageJ (http://imagej.nih.gov/ij/)
  • NOTE: The polarization of the excitation light can be controlled either by a liquid crystal–based universal polarizer or a filter wheel equipped with appropriately rotated linear polarizers (schematic of both present in Fig. A and B). OpenPolScope provides support for acquiring polarized fluorescence images using both of these modes. We first describe the use of the LC universal polarizer, which has certain advantages, as it can minimize polarization aberrations introduced by optical components in the light path. The filter wheel with polarizers, on the other hand, permits the use of broadband illumination light, even white light, while the LC universal polarizer requires narrowband monochromatic light of typically not more than 30 nm spectral bandwidth. The center wavelength, however, can vary between 450 and 700 nm for the LC universal polarizer.
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Figures

Videos

Literature Cited

Literature Cited
   DeMay, B.S. , Noda, N. , Gladfelter, A.S. , and Oldenbourg, R. 2011b. Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast. Biophys. J. 101:985‐994.
   DeMay, B.S. , Bai, X. , Howard, L. , Occhipinti, P. , Meseroll, R.A. , Spiliotis, E.T. , Oldenbourg, R. , and Gladfelter, A.S. 2011a. Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals. J. Cell Biol. 193:1065‐1081.
   Edelstein, A. , Amodaj, N. , Hoover, K. , Vale, R. , and Stuurman, N. 2010. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol. 14.20.11‐14.20.17.
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Internet Resources
   www.openpolscope.org
  Open‐access software for polarized light microscopy imaging and analysis.
   www.micro‐manager.org
  Open‐access imaging software for microscope automation (Edelstein et al., ).
   http://imagej.nih.gov/ij/
  Public domain image processing software.
   http://openpolscope.org/pages/Downloads.htm
  OpenPolScope protocol and downloads.
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