Optimizing Laser Source Operation for Confocal and Multiphoton Laser Scanning Microscopy

Gail McConnell1

1 University of Strathclyde, Glasgow, Scotland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.13
DOI:  10.1002/0471142956.cy0213s38
Online Posting Date:  November, 2006
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Confocal laser scanning microscopy (CLSM) and multiphoton laser scanning microscopy (MPLSM) are methods both widely used by life‐sciences researchers for imaging fluorescently labeled live cells and fixed tissue specimens. Key to the success of both CLSM and MPLSM is the application of a suitable laser source, namely one that provides sufficient average or peak power at the correct wavelength to excite fluorescence. High stability of the laser source output is required for three‐dimensional imaging, time‐lapse studies of live cells, and quantitative studies and inter‐experiment comparisons. The laser technology associated with the design of such lasers is mature, yet is unfortunately rather complex. This complexity can be off‐putting for the life‐sciences researcher who needs to optimize the system for the best possible images, but this apprehension can be overcome by understanding the function of the system components. This unit summarizes the optimization of the most commonly used laser sources for CLSM and MPLSM, including power and wavelength tuning and methods for cleaning optical components.

Keywords: lasers; krypton argon; Ti:Sapphire; laser diode; fluorescence; confocal microscopy; multi‐photon microscopy; laser scanning microscopy

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Table of Contents

  • Optimization of Laser Sources for CLSM
  • Optimization of Laser Sources for MPLSM
  • Cleaning Optical Elements
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Girkin, J.M. and McConnell, G. 2005. Advances in laser sources for confocal and multiphoton microscopy. Microsc. Res. Tech. 67:8‐14.
   Girkin, J.M., Ferguson, A.I., Wokosin, D.L., and Gurney, A.M. 2000. Confocal microscopy using an InGaN violet laser diode at 406 nm. Opt. Express 7:336‐341.
   Hell, S.W. and Stelzer, E.H.K. 1995. Lens aberrations in confocal fluorescence microscopy. In Handbook of Biological Confocal Microscopy (J. Pawley, ed.) pp. 347‐353. Plenum Press, N.Y.
   Long, L. and Hecht, J. 2005. Understanding Fiber Optics. Prentice Hall, Upper Saddle River, N.J.
   McConnell, G. 2004. Confocal laser scanning fluorescence microscopy with a visible continuum source. Opt. Express 12:2844‐2850.
   McConnell, G. 2005. Noise analysis of a white‐light supercontinuum light source for multiple wavelength confocal laser scanning fluorescence microscopy. J. Phys. D. Appl. Phys. 38:2620‐2624.
   McConnell, G. and Riis, E. 2004. Two‐photon laser scanning fluorescence microscopy using photonic crystal fiber. J. Biomed. Opt. 9:922‐927.
   Mueller, M., Squier, J., Wolleschensky, R., Simon, U., and Brakenhoff, G.J. 1998. Dispersion precompensation of 15 femtosecond optical pulses for high‐numerical‐aperture objectives. J. Microsc. 191:141‐150.
   Siegman, A.E. 1986. Lasers. University Science Books, Mill Valley, Calif.
   Spence, D.E., Kean, P.N., and Sibbett, W. 1991. 60‐fsec pulse generation from a self‐mode‐locked Ti:Sapphire laser. Opt. Lett. 16:42‐44.
   Thyagarajan, K. 1981. Laser Theory and Applications. Plenum Press, N.Y.
   White, N. and Errington, R. 2002. Multi‐photon microscopy: Seeing more by imaging less. Biotechniques 33:298‐300.
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