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Video-Rate Scanning Two-Photon Excitation Fluorescence Microscopy

Published online by Cambridge University Press:  02 July 2020

G.Y. Fan ,
H. Fujisaki ,
R.-K. Tsay ,
R.Y. Tsien  and
Mark H. Ellisman
G.Y. Fan
Affiliation:
National Center for Microscopy and Imaging Research, Dept. of Neurosciences, San Diego, La Jolla, CA, 92093
H. Fujisaki
Affiliation:
Nikon Corp., Tsukuba Research Lab., 5-9-1 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
R.-K. Tsay
Affiliation:
National Center for Microscopy and Imaging Research, Dept. of Neurosciences, San Diego, La Jolla, CA, 92093
R.Y. Tsien
Affiliation:
Howard Hughes Medical Institute, Dept. of Chemistry and Biochemistry,University of California, San Diego, La Jolla, CA, 92093
Mark H. Ellisman
Affiliation:
National Center for Microscopy and Imaging Research, Dept. of Neurosciences, San Diego, La Jolla, CA, 92093
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Extract

A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.

Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.

Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.

Type
New Developments in Multi-Photon Excitation Microscopy
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 424 - 425
Copyright
Copyright © Microscopy Society of America

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References

1.Denk, W., Strickler, J.H. and Webb, W.W., Science 248 (1990) 73.CrossRefGoogle Scholar
2.Tsien, R.Y. and Bacskai, B.J., In Pawley, J.B., Ed., Handbook of biological confocal microscopy, 2nd Ed., New York Plenum (1995) 459.CrossRefGoogle Scholar
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4. This work is supported in part by NIH grants NS14718, NS26739 and RR04050.Google Scholar