© 2003 The Royal Microscopical Society Journal of Microscopy, Vol. 211, Pt 3 September 2003, pp. 191–207 Received 16 October 2002; accepted 19 May 2003 Blackwell Publishing Ltd. INVITED REVIEW Quantitative fluorescent speckle microscopy: where it came from and where it is going G. DANUSER & C. M. WATERMAN-STORER* BioMicrometrics Group, Laboratory for Biomechanics, ETH Zürich, 8952 Schlieren, Switzerland *Department of Cell Biology and Institute for Childhood and Neglected Diseases, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, U.S.A. Key words. Cytoskeleton, fluorescent speckle microscopy, polymer. Summary Fluorescent speckle microscopy (FSM) is a technology for analysing the dynamics of macromolecular assemblies. Origi- nally, the effect of random speckle formation was discovered with microtubules. Since then, the method has been expanded to other proteins of the cytoskeleton such as f-actin and microtubule binding proteins. Newly developed, specialized software for analysing speckle movement and photometric fluctuation in the context of polymer transport and turnover has turned FSM into a powerful method for the study of cytoskeletal dynamics in cell migration, division, morphogen- esis and neuronal path finding. In all these settings, FSM serves as the quantitative readout to link molecular and genetic interventions to complete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering of molecular regulation of the cytoskeleton. Fully automated FSM assays can also be applied to live-cell screens for toxins, chemicals, drugs and genes that affect cytoskeletal dynamics. We envision that FSM has the potential to become a core tool in automated, cell-based molecular diagnostics in cases where variations in cytoskeletal dynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. In this paper, we review the origins of FSM, discuss these most recent technical developments and give a glimpse to future directions and potentials of FSM. It is written as a complement to the recent review (Waterman-Storer & Danuser, 2002, Curr. Biol., 12, R633–R640), in which we emphasized the use of FSM in cell biological applications. Here, we focus on the technical aspects of making FSM a quantitative method. Received 16 October 2002; accepted 19 May 2003 Introduction Fluorescent speckle microscopy (FSM) is a method to analyse the movement, assembly and disassembly dynamics of macro- molecular structures in vivo and in vitro (Waterman-Storer et al., 1998). As reviewed in Waterman-Storer & Danuser (2002), FSM capitalizes on the well-established method of fluorescent analogue cytochemistry, in which purified protein is covalently linked to a fluorophore and microinjected or expressed as a green fluorescent protein (GFP) fusion in living cells, incorporated into cellular structures, and visualized by wide-field epifluorescence microscopy (Wang et al., 1982; Prasher, 1995). This classic approach has yielded much information about protein localization and macromolecular structure dynamics in cells, but has been limited in its ability to report protein dynamics because of inherently high back- ground fluorescence from unincorporated and out-of-focus incorporated fluorescent subunits and the difficulty in detect- ing movement or turnover of subunits because of the uniform labelling of fluorescent structures. These problems have been partially alleviated by use of technically cumbersome laser photo-bleaching and photo-activation of fluorescence to mark structures in limited cell areas and measure the movement of subunit turnover in this area at steady state (Wang, 1985; Wadsworth & Salmon, 1986; Mitchison, 1989; Wolf, 1989; Theriot & Mitchison, 1991). Similar to these techniques is the recently introduced ratiometric method of fluorescence locali- zation after photobleaching (FLAP; Dunn et al., 2002; Zicha et al., 2003). FSM provides similar information to all of these photo-marking techniques. However, it delivers simultaneous kinetic information in large areas of the cell, offering the ability to detect non-steady state dynamics of molecular systems at high spatial and temporal resolution. In addition, FSM significantly reduces out-of-focus fluorescence and improves the visibility of fluorescently labelled structures and their dynamics in three-dimensional (3D) polymer arrays Correspondence: Gaudenz Danuser, Laboratory for Computational Cell Biol- ogy, Department of Cell Biology, CB 167, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA. Tel: +1 858 784 7096; fax: +1 858 784 9779; e-mail: gdanuser@scripps.edu