Noninvasive chemical analysis of biological systems based on advanced coherent Raman microspectroscopy
Final Report Abstract
This continuation project has focused on the further development and the demonstration of compelling applications of two quantitative coherent Raman scattering microscopy techniques: While hyperspectral coherent anti-Stokes Raman scattering (CARS) imaging provides fast access to the full wealth of the sample’s Raman spectral response, vibrational imaging based on high-sensitivity stimulated Raman scattering (SRS) microscopy allows the fast point-by-point 3D chemical mapping of a sample at a selected Raman mode in a quantitative manner. We have experimentally implemented hyperspectral CARS imaging with fast acquisition times in the order of milliseconds per pixel based on ultrabroadband Stokes continuum pulses generated in a photonic crystal fiber, which allows for the simultaneous detection of the vibrational modes residing in the C-H (lipid) and the fingerprint (protein) regions, covering a range from 300 cm-1 to 4000 cm-1. Furthermore, we have developed the principle concept of hyperspectral CARS imaging, involving (i) the recording of the hyperspectral CARS data cube, (ii) retrieving its spectral phases, followed by (iii) the quantitative spectral analysis using univariate or multivariate methods. To illustrate the potential of this concept for the noninvasive chemical analysis of biological systems without a priori knowledge, we have studied the lipid uptake mechanism into human macrophages as a function of different atherogenic model lipoproteins, and demonstrated the capability of quantitative in-vivo chemical structure analysis of intra-organelle lipids by mapping their distinct degrees of acyl chain unsaturation. This information content cannot be obtained by conventional biomedical microscopy techniques in a noninvasive manner. The yet-to-be-published work described here has been carried out in close collaboration with the group of Prof. G. Schmitz at the Universitätsklinikum Regensburg. In parallel, we have developed our second-generation SRS microscope using near-transformlimited, picosecond pulses at a repetition rate of ~76 MHz and amplitude-modulation of the Stokes (or pump) pulse train intensity at radio-frequencies (MHz) where laser noise is negligible in conjunction with phase-sensitive lock-in detection. This way, we have succeeded in the first SRS imaging with high detection sensitivity, which is intrinsically free of nonlinear nonresonant background, and allows the direct and quantitative interpretation of the sample’s vibrational image contrast. Furthermore, we have demonstrated the potential of SRS imaging for live-cell imaging. This technique has been identified as a powerful, noninvasive tool for biomedical imaging applications, and has attracted high attention, for example, in news feature coverage in Nature, 459 (4th June 2009) and the Best of 2009 Papers Award in New J. Phys. for its presentation of outstanding new research. Our research results demonstrate the high potential of coherent Raman microscopy as a complementary and noninvasive tool for further compelling applications in the Life Sciences, which circumvents current limitations in fluorescent-based microscopy approaches, and offer the basis for the further development of our research program.
Publications
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Intracellular coherent anti-Stokes Raman scattering (CARS) microspectroscopy. Biophys. J. Suppl. S (2007) 196A
A. Kovalev, N. Patincharath, A. Volkmer
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Quantitative analysis of coherent anti- Stokes Raman scattering (CARS) spectra from a single collagen fibril. Biophys. J. Suppl. S (2007) 161A
S. Busch S, A. Muschielok, A. Kovalev, A. Volkmer
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CARS microspectroscopy: exploring the chemical and physical structure of individual biopolymers, living cells, and tissue. SPIE BIOS 2009 Conf. 7183 (2009) 7183-23
A. Kovalev and A.Volkmer
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Coherent Raman scattering microscopy. In: Emerging Biomedical and Pharmaceutical Applications of Raman Spectroscopy. Eds. P. Matousek and M. Morris, 2009 (Springer-Verlag, Berlin, Heidelberg, New York, Tokyo, ISBN: 978-3-642-02648-5)
A. Volkmer
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Labelfreie Bildgebung mittels kohärenter Raman-Streunung. Laborwelt 10(1) (2009) 12-16
A. Volkmer
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New trends and recent advances in coherent Raman microscopy and nonlinear optical spectrsocopy: introduction to the special issue. J. Raman Spectrosc., 40 (2009) 712
A. Volkmer, P. P. Radi, A. M. Zheltikov, A. Zumbusch
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Vibrational Imaging based on stimulated Raman scattering microscopy. New J. Phys., 11 (2009) 033026
P. Nandakumar, A. Kovalev, A. Volkmer
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Kohärente Raman-Mikroskopie. Physik Journal 10(3) (2011) 71
A. Volkmer, A. Zumbusch und Y. Silberberg
