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 sensitivity of Raman based label-free chemical imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. Based on 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 mixed chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, an entire Raman spectral data cube can be obtained within a minute. We show that mammalian cells 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.

ABL1 tyrosine-kinase inhibitors (TKI) are a front-line therapy for chronic myelogenous leukemia and represent the best known examples of targeted cancer therapeutics. However, the dynamic uptake of low molecular weight TKIs into cells and their intracellular behavior is largely unknown due to the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs – imatinib and nilotinib inside living cells using hyperspectral stimulated Raman scattering imaging. Both drugs were enriched over 1000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced by more than 10-fold when using chloroquine simultaneously, suggesting that chloroquine may increase the efficacy of TKIs through lysosome mediated drug-drug interaction besides the commonly proposed autophagy inhibition mechanism.

Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a non-destructive, label-free optical method, to reveal glioma infiltration in animal models. Here we show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ=0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density and protein:lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density and protein:lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Importantly, quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

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

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Metabolic fingerprinting provides valuable information on the physiopathological states of cells and tissues. Traditional imaging mass spectrometry and magnetic resonance imaging are unable to probe the spatial-temporal dynamics of metabolites at the subcellular level due to either lack of spatial resolution or inability to perform live cell imaging. Here we report a complementary metabolic imaging technique that is based on hyperspectral stimulated Raman scattering (hsSRS). We demonstrated the use of hsSRS imaging in quantifying two major neutral lipids: cholesteryl ester and triacylglycerol in cells and tissues. Our imaging results revealed previously unknown changes of lipid composition associated with obesity and steatohepatitis. We further used stable-isotope labeling to trace the metabolic dynamics of fatty acids in live cells and live Caenorhabditis elegans with hsSRS imaging. We found that unsaturated fatty acid has preferential uptake into lipid storage while saturated fatty acid exhibits toxicity in hepatic cells. Simultaneous metabolic fingerprinting of deuterium-labeled saturated and unsaturated fatty acids in living C. elegans revealed that there is a lack of interaction between the two, unlike previously hypothesized. Our findings provide new approaches for metabolic tracing of neutral lipids and their precursors in living cells and organisms, and could potentially serve as a general approach for metabolic fingerprinting of other metabolites.

Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Here we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.

We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10(-6) absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.