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A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells

Published online by Cambridge University Press:  02 July 2020

Paul Maddox ,
Julie Canman ,
Sonia Grego ,
Wendy Salmon ,
Clare Waterman-Storer  and
E.D. Salmon
Paul Maddox
Affiliation:
Department of Biology, University of North Carolina, Chapel Hill, NC27599-3280
Julie Canman
Affiliation:
Department of Biology, University of North Carolina, Chapel Hill, NC27599-3280
Sonia Grego
Affiliation:
Department of Biology, University of North Carolina, Chapel Hill, NC27599-3280
Wendy Salmon
Affiliation:
Department of Biology, University of North Carolina, Chapel Hill, NC27599-3280
Clare Waterman-Storer
Affiliation:
Department of Cell Biology, Scripps Research Institute, La Jolla, CA92037
E.D. Salmon
Affiliation:
Department of Cell Biology, Scripps Research Institute, La Jolla, CA92037
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Abstract

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.

Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.

Type
Video and Digital Imaging Microscopy: a Symposium Honoring Dr. Shinya Inoue (Organized by E. Salmon)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 8 - 9
Copyright
Copyright © Microscopy Society of America 2001

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References

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