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Special Section on Coherent Raman Imaging Techniques and Biomedical Applications

Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples

[+] Author Affiliations
Christoph Pohling, Tiago Buckup, Marcus Motzkus

Physikalisch-Chemisches Institut, Ruprecht-Karls-Universität, Im Neuenheimer Feld 229, D-69120 Heidelberg, Germany

J. Biomed. Opt. 16(2), 021105 (February 16, 2011). doi:10.1117/1.3533309
History: Received June 30, 2010; Revised August 18, 2010; Accepted August 23, 2010; Published February 16, 2011; Online February 16, 2011
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Multiplex coherent anti-Stokes Raman scattering (MCARS) provides labeling free and fast characterization of materials and biological samples in nonlinear microscopy. In spite of its success, remaining challenges regarding the data analysis for chemoselective imaging still have to be solved. In general, image contrast has been realized by using only one spectral feature directly taken from the unprocessed raw data. This procedure is limited to strong and well separated Raman resonances like the saturated CH-stretching vibration of lipids in the case of biological samples. In order to overcome this limitation, we present a new method of MCARS data processing that exploits the whole measured spectrum to disentangle overlapping contributions of different (bio-) chemical components. Our “two-step” approach is based on the combination of imaginary part extraction followed by global fitting of the hyperspectral data set. Previous knowledge about the sample, e.g., pure spectra of the individual components is no longer necessary. The result is a highly contrasted image, where the patterns and differences between the sample components can be represented in different colors. We successfully applied this method to complex structured polymer samples and biological tissues.

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© 2011 Society of Photo-Optical Instrumentation Engineers (SPIE)

Citation

Christoph Pohling ; Tiago Buckup and Marcus Motzkus
"Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples", J. Biomed. Opt. 16(2), 021105 (February 16, 2011). ; http://dx.doi.org/10.1117/1.3533309


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