A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells.
Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.
Adams
Michael C
MC
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, 92037, La Jolla, CA, USA.
Salmon
Wendy C
WC
Gupton
Stephanie L
SL
Cohan
Christopher S
CS
Wittmann
Torsten
T
Prigozhina
Natalie
N
Waterman-Storer
Clare M
CM
D000818:Animals; D002465:Cell Movement; D003599:Cytoskeleton / Q000502:physiology; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure