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: 12.11). 02/2012; 134(8):3623-6. DOI: 10.1021/ja210081h
Source: PubMed


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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    • "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: To date, medical imaging of tissues has largely relied on time-consuming staining processes, and there is a need for rapid, label-free imaging techniques. Stimulated Raman scattering microscopy offers a three-dimensional, real-time imaging capability with chemical specificity. However, it can be difficult to differentiate between several constituents in tissues because their spectral characteristics can overlap. Furthermore, imaging speeds in previous multispectral stimulated Raman scattering imaging techniques were limited. Here, we demonstrate label-free imaging of tissues by 30 frames/s stimulated Raman scattering microscopy with frame-by-frame wavelength tunability. To produce multicolour images showing different constituents, spectral images were processed by modified independent component analysis, which can extract small differences in spectral features. We present various imaging modalities such as two-dimensional spectral imaging of rat liver, two-colour three-dimensional imaging of a vessel in rat liver, spectral imaging of several sections of intestinal villi in mouse, and in vivo spectral imaging of mouse ear skin.
    Nature Photonics 11/2012; 6(12). DOI:10.1038/NPHOTON.2012.263 · 32.39 Impact Factor
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    • "Algal culturing using Coccomyxa subellipsoidea (Coccomyxa sp.) C169 was carried out in this study. Cultures used for inoculum were maintained on agar plates containing Bold’s basal medium (BBM) [33] with double the normal nitrate concentration and 100 mg L−1 carbenicillin (Fisher Scientific, Pittsburg, PA). Liquid batch cultures were first established in BBM from picked colonies and allowed to reach the early stationary phase at 25°C and 120 rpm in a lighted shaking incubator (Innova 43, New Brunswick Scientific, Enfield, CT). "
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    ABSTRACT: Microalgae are extensively researched as potential feedstocks for biofuel production. Energy-rich compounds in microalgae, such as lipids, require efficient characterization techniques to investigate the metabolic pathways and the environmental factors influencing their accumulation. The model green alga Coccomyxa accumulates significant amounts of triacylglycerols (TAGs) under nitrogen depletion (N-depletion). To monitor the growth of TAGs (lipid) in microalgal cells, a study of microalgal cells (Coccomyxa sp. C169) using both spontaneous Raman and coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy were carried out. Spontaneous Raman spectroscopy was conducted to analyze the components in the algal cells, while CARS was carried out to monitor the distribution of lipid droplets in the cells. Raman signals of carotenoid are greater in control microalgae compared to N-depleted cells. Raman signals of lipid droplets appear after N-depletion and its distribution can be clearly observed in the CARS microscopy. Both spontaneous Raman spectroscopy and CARS microscopy were found to be suitable analysis tools for microalgae.
    Biomedical Optics Express 11/2012; 3(11):2896-906. DOI:10.1364/BOE.3.002896 · 3.65 Impact Factor
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    ABSTRACT: We propose the collinear balanced detection (CBD) technique for noise suppression in fiber laser (FL)-based stimulated Raman scattering (SRS) microscopy. This technique reduces the effect of laser intensity noise at a specific frequency by means of pulse splitting and recombination with a time delay difference. We experimentally confirm that CBD can suppress the intensity noise of second harmonic (SH) of Er-FL pulses by 13 dB.The measured noise level including the thermal noise is higher by only ~1.4 dB than the shot noise limit. To demonstrate SRS imaging, we use 4-ps SH pulses and 3-ps Yb-FL pulses, which are synchronized subharmonically with a jitter of 227 fs. The effectiveness of the CBD technique is confirmed through SRS imaging of a cultured HeLa cell.
    Optics Express 06/2012; 20(13):13958-65. DOI:10.1364/OE.20.013958 · 3.49 Impact Factor
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