26] Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells

Abstract

Publisher Summary This chapter describes a spinning disk confocal fluorescence microscope system that is initially assembled for fluorescent speckle microscopy (FSM) of the assembly dynamics and movements of individual microtubules and actin filament arrays within cells. The random nature of subunit association creates a nonuniform fluorescent “speckle” pattern along the polymer lattice during polymerization. FSM can also be used to record the binding and release of microtubule associated proteins (MAPs) on the microtubule lattice. Optimum contrast is obtained for fuorescent speckles containing only a few fluorophores, often five or fewer, within the maximum resolution limits of the light microscope. In addition to applications in FSM, the resolution and sensitivity of this instrument have proved valuable for live cell imaging of green fluorescent protein (GFP) fusion proteins. Also, the high signal-to-noise ratio of images obtained with this instrument has provided the opportunity to obtain three-dimensional (3-D) immunofluorescent images of extraordinary resolution and image quality by constrained iterative deconvolution.