Small molecules such as metabolites and drugs are crucial to the biochemistry of living organisms because of their large diversity and the sheer number present. Unlike their large molecule counter parts such proteins, DNA and mRNA, most of these species are invisible in a living cell or organism because of technical difficulties. Tagging them with bulky fluorescent labels perturbs their function. Label-free optical imaging based on Raman scattering is highly desirable because it offers contrast based on the intrinsic chemical properties of molecules via molecular vibrational spectroscopy, but has been limited to low sensitivity and long acquisition times. Our group has recently developed stimulated Raman scattering (SRS) microscopy, which offers an unprecedented combination of high sensitivity, rapid image acquisition, chemical specificity and noninvasiveness. The development of SRS microscopy has generated a great of interest from the biomedical research community and studies of lipid metabolism and drug distributions in tissue are already underway in our laboratory. We propose to continue the pioneering development in order to probe lipid metabolism, including lipogenesis, lipolysis and insulin resistance. We will image not only different types of lipids, such as saturated and unsaturated lipids and cholesterol, but also key metabolites, such as glucose, ATP, ADP, and small molecule drugs as well, all in living cells and tissue without the use of perturbative labels. These new experiments will no doubt allow new observations that will enrich our knowledge of cellular biochemistry. We will also develop all-optical cancer markers and use SRS to bring about a new paradigm for intra-operative imaging of brain tumors, improving the surgical outcome by shortening surgery and allowing more precise definition of tumor margins. The widespread adoption of SRS and the discovery of new applications will revolutionize many areas of biomedical research, including lipid metabolism, pharmacokinetics and cancer diagnosis.

Public Health Relevance

The ability to visualize small molecules such as metabolites and drugs in living cells and organisms without labels will revolutionize biomedical research, particularly lipid metabolism, pharmacokinetics and cancer diagnosis. We propose to extend the recently developed stimulated Raman scattering microscopy technique to offer rapid, sensitive and noninvasive imaging based on intrinsic vibrational frequencies of molecules, in order to selectively map the distributions of lipids, protein, glucose, cholesterol, ADP, ATP, folate and small molecule drugs, among other species. This technology will make an unprecedented transformation of our ability to monitor biochemistry in real time in living systems, and lead to a potential breakthrough in intra-operative imaging for rapid and precise tumor identification.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-A (51))
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Conroy, Richard
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Harvard University
Schools of Arts and Sciences
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Fu, Dan; Yu, Yong; Folick, Andrew et al. (2014) In vivo metabolic fingerprinting of neutral lipids with hyperspectral stimulated Raman scattering microscopy. J Am Chem Soc 136:8820-8
Fu, Dan; Zhou, Jing; Zhu, Wenjing Suzanne et al. (2014) Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering. Nat Chem 6:614-22
Zhao, Ziqing W; Roy, Rahul; Gebhardt, J Christof M et al. (2014) Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc Natl Acad Sci U S A 111:681-6
Freudiger, Christian W; Yang, Wenlong; Holtom, Gary R et al. (2014) Stimulated Raman Scattering Microscopy with a Robust Fibre Laser Source. Nat Photonics 8:153-159
Fu, Dan; Holtom, Gary; Freudiger, Christian et al. (2013) Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers. J Phys Chem B 117:4634-40
Lamb, Erin S; Lefrancois, Simon; Ji, Minbiao et al. (2013) Fiber optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy. Opt Lett 38:4154-7
Kong, Lingjie; Ji, Minbiao; Holtom, Gary R et al. (2013) Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator. Opt Lett 38:145-7
Gebhardt, J Christof M; Suter, David M; Roy, Rahul et al. (2013) Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat Methods 10:421-6
Ji, Minbiao; Orringer, Daniel A; Freudiger, Christian W et al. (2013) Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med 5:201ra119
Fu, Dan; Lu, Fa-Ke; Zhang, Xu et al. (2012) Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. J Am Chem Soc 134:3623-6

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