Unit

UNIT 2.18 Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics

  1. Michelle C. Mendoza,
  2. Sebastien Besson,
  3. Gaudenz Danuser

Published Online: 1 OCT 2012

DOI: 10.1002/0471142956.cy0218s62

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Mendoza, M. C., Besson, S. and Danuser, G. 2012. Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics. Current Protocols in Cytometry. 62:2.18:2.18.1–2.18.26.

Author Information

  1. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 OCT 2012
  2. Published Print: OCT 2012

Abstract

Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. Curr. Protoc. Cytom. 62:2.18.1-2.18.26. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • live-cell imaging;
  • microinjection;
  • image processing;
  • actin;
  • speckles