Article

Hyperspectral Imaging with Stimulated Raman Scattering by Chirped Femtosecond Lasers

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States
J. Phys. Chem. B, 2013, 117 (16), pp 4634–4640
DOI: 10.1021/jp308938t
Publication Date (Web): December 20, 2012
Copyright © 2012 American Chemical Society
This article is part of the Paul F. Barbara Memorial Issue special issue.

Abstract

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Raman microscopy is a quantitative, label-free, and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman scattering (SRS) microscopy have significantly increased the acquisition speed of Raman based label-free imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. On the basis of a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of cholesterol in the presence of interfering chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, hyperspectral imaging data in the C–H stretching region is obtained within a minute. We show that mammalian cell SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol, and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.

Detailed descriptions of experimental setup, data analysis, and high resolution imaging results. This material is available free of charge via the Internet at http://pubs.acs.org.

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Article Views: 2,323 Times
Received 8 September 2012
Published online 20 December 2012
Published in print 25 April 2013
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