Fluorescent speckle microscopy.

INTRODUCTION: Fluorescent speckle microscopy (FSM) is a live imaging and quantitative measurement technique used for analyzing motion and turnover of macromolecular assemblies in vivo and in vitro. It differs from related imaging techniques such as photobleaching and photoactivation in its use of substantially lower concentrations of fluorescently labeled assembly subunits. When small numbers of labeled subunits and large numbers of unlabeled subunits become randomly incorporated together into a macromolecular structure, the random distribution of fluorophores generates nonuniform fluorescence intensity patterns that appear as distinct puncta against low background fluorescence. These puncta, called speckles, serve as fiduciary markers so that motion and turnover of the structure are visualized. Computational analysis of speckle image data transforms FSM into a powerful tool for high-resolution quantitative analysis of macromolecular assembly dynamics. Successful application of FSM depends on the ability to reliably generate and image speckles, which are characterized by their weak emission signals, and to effectively extract quantitative information through computational analysis of speckle image data, which are characterized by their stochastic fluctuations, low signal-to-noise ratios, and high spatiotemporal complexity. This article aims to provide a practical introduction to basic principles, experimental implementation, and computational data analysis of FSM. Examples are used to show the application of FSM in analyzing the dynamic organization and assembly/disassembly of cytoskeletal filament networks, an area in which FSM analysis has found great success.