UNIT 2.18 Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics
Published Online: 1 OCT 2012
Copyright © 2012 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 OCT 2012
- Published Print: OCT 2012
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.
- live-cell imaging;
- image processing;