Spinning Disk Confocal Microscopy
Spinning disk microscopy has advanced significantly in the past decade and now represents one of the optimum solutions for both routine and high-performance live-cell imaging applications. The rapid expansion in biomedical research using live-cell imaging techniques over the past several years has been fueled by a combination of events that include dramatic advances in spinning disk confocal microscopy instrumentation coupled with the introduction of novel ultra-sensitive detectors and continued improvements in the performance of genetically-encoded fluorescent proteins.
Introduction to Spinning Disk Confocal Microscopy - Acquiring images of localized fluorophores in living cells on the millisecond timescales that reveal intricate biological dynamics presents a host of new challenges, which are far more complex than the traditional issues associated with creating a single high-resolution snapshot of well-stained fixed tissue in a laser scanning confocal microscope. Review spinning disk microscopy from a historical perspective, as well as current instrumentation that is being used today. Also included is information on resolution and digital camera systems.
Interactive Flash Tutorials
Spinning Disk Fundamentals - Explore how light passes through the pinholes on a spinning disk microscope to produce multiple excitation beams that are swept across the specimen as the disk spins. The Nipkow disk is located in a conjugate image plane and scans with approximately 1000 individual light beams.
Yokogawa Spinning Disk Scanning Unit - The most advanced design in spinning disk instruments was engineered by Yokogawa Electric Corporation of Japan and implemented in a series of increasingly complex disk scanning units. This tutorial examines the operating principles of the Yokogawa scanning units.
Pinhole Crosstalk in Spinning Disk Microscopy - Axial resolution in spinning disk microscopy is largely defined by the size of the pinhole or slit and the separation distances between these apertures. This tutorial demonstrates how fluorescence removed from the focal plane can generate pinhole crosstalk.
Microlens Arrays in Spinning Disk Microscopy - The amount of light transmitted through the Nipkow disk in spinning disk microscopy is determined by the diameter of the pinhole or slit and the distance between these apertures. This tutorial explores how the amount of light passed through a disk can be increased by using microlens arrays on the upper disk in a two-disk system.
References and Resources
Spinning Disk Confocal Microscopy - Spinning disk confocal microscopy is rapidly emerging as the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems are capable of providing high acquisition speeds with acceptable contrast and minimal photobleaching at the low light levels available with this technique. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spinning disk confocal microscopy.
Live-Cell Imaging - FRET with Spectral Imaging and Linear Unmixing - The introduction of genetically-encoded fluorescent protein fusions as a localization marker in living cells has revolutionized the field of cell biology, and the application of photostable quantum dots looms on the horizon. Live-cell imaging techniques now involved a wide spectrum of imaging modalities, including widefield fluorescence, confocal, multiphoton, total internal reflection, FRET, lifetime imaging, superresolution, and transmitted light microscopy. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of live-cell imaging.
Fluorescent Proteins - The growing class of fluorescent proteins useful for detecting events in living cells and animals has almost single-handedly launched and fueled a new era in biology and medicine. These powerful research tools have provided investigators with a mechanism of fusing a genetically encoded optical probe to a practically unlimited variety of protein targets in order to examine living systems using fluorescence microscopy and related technology. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of fluorescent protein technology.
Derek K. Toomre - Department of Cell Biology, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut, 06520.
Matthias F. Langhorst - Carl Zeiss MicroImaging GmbH, Koenigsallee 9-21, 37081 Goettingen, Germany.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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