Hostname: page-component-cd9895bd7-mkpzs Total loading time: 0 Render date: 2024-12-23T00:20:10.207Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Get access

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1. Waterman-Storer, C.M. and Salmon, E. D.. Biophysical J. 75(1998)2059.CrossRefGoogle Scholar

2. Waterman-Storer, C. M. et al., in Current Biol. 8(1998)1227.CrossRefGoogle Scholar

3. Waterman-Storer, C. M. and Salmon, E. D., in FASEB J. 13(1999)S225.CrossRefGoogle Scholar

4. Maddox, P. et al., in Nature Cell Biol. 2(2000)36.CrossRefGoogle Scholar

5. Shaw, S. L. et al., in Current Biol. 7(1997)701.CrossRefGoogle Scholar

6. Inoue, S. and Inoue, T., in Meth. in Cell Biol. 38(2000) Chapter 5.Google Scholar

7. Agard, D. A. et al., in Meth. in Cell Biol. 30(1989)353.CrossRefGoogle Scholar

8. Inoue, S. and Salmon, E. D.. Mol. Biol. of the Cell. 6(1995)1619.CrossRefGoogle Scholar