Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Journal of the American Chemical Society (Impact Factor: 11.44). 02/2012; 134(8):3623-6. DOI: 10.1021/ja210081h
Source: PubMed

ABSTRACT Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.

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Available from: Gary Holtom, Jul 10, 2014
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    • "Femtosecond SRS microscopy [15] can acquire Raman spectra with a spectrometer, but requires intense excitation pulses, which are not compatible with biological imaging. Another approach for pushing the chemical specificity is SRS spectral imaging [16-18, Fu, D. et al., Photonics West 2012, paper 8226-58 (2012)], where SRS images at various vibrational frequencies are successively acquired. This may allow us to analyze vibrational spectrum so as to specifically detect molecules. "
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    ABSTRACT: Stimulated Raman scattering (SRS) microscopy has opened up a wide range of biochemical imaging applications by probing a particular Raman-active molecule vibrational mode in the specimen. However, the original implementation with picosecond pulse excitation can only realize rapid chemical mapping with a single Raman band. Here we present a novel SRS microscopic technique using a grating-based pulse shaper for excitation and a grating-based spectrograph for detection to achieve simultaneous multicolor SRS imaging with high sensitivity and high acquisition speeds. In particular, we used linear combination of the measured CH2 and CH3 stretching signals to map the distributions of protein and lipid contents simultaneously.
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