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Proceedings Paper

Application of coherent antistokes Raman scattering (CARS) to imaging mammalian cells: a means for gaining molecular selectivity in multiphoton imaging
Author(s): Gary R. Holtom; Brian Thrall; Thomas Weber; Leyun Zhu; Derek Hopkins; Christopher Parkinson; Steven Colson; John M. Price; Beek Yoke Chin; Augustine Choi; Terence Risby
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Paper Abstract

Virtually all laser based microscopy imaging methods involve a single laser, with ultrafast lasers emerging as the enabling tool for a variety of methods. Two-photon fluorescence is a high sensitivity method with selectivity depending on a chromophore that is either added or produced by genetic engineering. While there are fundamental advantages over white light or other fluorescence microscopies, there are unavoidable limitations such as bleaching, photoinduced damage to the cell, and the inability to label some major constituents of the cell, particularly the abundant species. Raman imaging affords chemical selectivity but application is limited due particularly to its low sensitivity and unavoidable fluorescence background. Adding a second laser beam, shifted from the first laser by a molecular vibrational frequency, increases the detected Raman signal by many orders of magnitude and in addition shifts the detected signal to the high energy (blue) side of both lasers, removing fluorescence artifacts. Signal levels sufficient to acquire high signal-to-noise ratio images of 200 by 200 pixels in one minute requires sub-nanojoule pulse energy. A convenient, tunable source of the Stokes-shifted beam is provided by an Optical Parametric Amplifier (OPA), which requires an amplified laser. 250-kHz sources have ample energy and in addition keep the average sample power on the order of 0.1 mW, a level that even sensitive biological systems tolerate at the focal spot diameter of 0.3 micrometers . Long-term viability of mammalian cells has been demonstrated during dozens of scans in a single plane. Two-photon fluorescence provides a useful complimentary data channel that is acquired simultaneously with the Raman image. Several dyes and green fluorescence protein have been used for this purpose. Interpretation of images, acquiring three dimensional images, and identification of cellular features are ongoing activities.

Paper Details

Date Published: 24 April 2001
PDF: 10 pages
Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); doi: 10.1117/12.424571
Show Author Affiliations
Gary R. Holtom, Pacific Northwest National Lab. (United States)
Brian Thrall, Pacific Northwest National Lab. (United States)
Thomas Weber, Pacific Northwest National Lab. (United States)
Leyun Zhu, Pacific Northwest National Lab. (United States)
Derek Hopkins, Pacific Northwest National Lab. (United States)
Christopher Parkinson, Pacific Northwest National Lab. (United States)
Steven Colson, Pacific Northwest National Lab. (United States)
John M. Price, Pacific Northwest National Lab. (United States)
Beek Yoke Chin, Johns Hopkins Univ. (United States)
Augustine Choi, Univ. of Pittsburgh (United States)
Terence Risby, Johns Hopkins Univ. (United States)

Published in SPIE Proceedings Vol. 4262:
Multiphoton Microscopy in the Biomedical Sciences
Ammasi Periasamy; Peter T. C. So, Editor(s)

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