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High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)

Published online by Cambridge University Press:  02 July 2020

P. W. Wiseman ,
J. C. Bouwer ,
S. Peltier  and
M. H. Ellisman
P. W. Wiseman
Affiliation:
Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, Ca, 92093-0339, USA
J. C. Bouwer
Affiliation:
Dept of Neuroscience, University of California, San Diego, La Jolla, Ca, 92093-0608, USA
S. Peltier
Affiliation:
Dept of Neuroscience, University of California, San Diego, La Jolla, Ca, 92093-0608, USA
M. H. Ellisman
Affiliation:
Dept of Neuroscience, University of California, San Diego, La Jolla, Ca, 92093-0608, USA
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Abstract

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.

We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

Type
Living Microscopy: from Cells to Whole Animals (Organized by D. Piston)
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. 22 - 23
Copyright
Copyright © Microscopy Society of America 2001

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References

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5. This work is supported grant NIH NCRR RR04050 to M. EllismanGoogle Scholar