Coherent Raman Scattering (CRS) Microscopy
First Fully-Integrated Stimulated Raman Scattering (SRS) Microscope
INVENIO presents the first fully-integrated SRS R&D microscope based on an all-fiber laser source. Seamless integration of the laser source and the microscope through fiber delivery increases long-term stability and ease of use. In the current implementation, the Raman coverage is from 2800 cm-1 to 3100 cm-1.The system has been tailored for multimodal, label-free tissue imaging applications and offers a 4-channel epi-PMT detector for simultaneous acquisition of CARS and other multi-photon signals, such as two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) alongside with transmission SRS.
(for Research Use only)
Wavelength 1: 790 nm (~150 mW)
Wavelength 2: 1015-1045 nm
Raman coverage: 2800 - 3100 cm-1; wavelength extension unit for 2000-2200 cm-1 region (in development)
Pulse duration: 2-3 ps
Timing jitter: <15 fs (between the two excitation wavelengths)
Repetition rate: 80 MHz
Stand: Olympus BX43
Stage: Motorized XYZ-stage
Optics: Single Olympus objective
o Magnifications: 20x - 100x
o Numerical aperture: 0.75 - 1.2NA
o Immersion: water
Imaging Speed: 0.1 – 4 frames/s with sampling up to 4096 x 4096 (with non-resonant scan mirrors
o Up to 4 simultaneous epi-PMTs
o 1 transmission-SRS detector
o 1 epi-SRS detector
Imaging software: MicroManager
Examples in Lipid Biology R&D
"Lipids droplets are ubiquitous cellular organelles that store and regulate lipids for energy metabolism. Their quantity, localization, and composition are intimately associated with metabolic diseases such as diabetes. One challenge in understanding the functional roles of lipid droplets in the pathophysiology of tissue is a lack of noninvasive and quantitative tools that can directly image lipid droplets at high spatial and temporal resolution. Hyperspectral SRS imaging overcomes this challenge by directly probing the vibrational signature of abundant C-H bonds within lipids, without resorting to lipid staining. Importantly, different lipids can be distinguished based on their variations in vibrational spectra. In combination with deuterated tracing, which can monitoring lipid synthesis, degradation, and conversion in real-time at the single droplets level, hyperspectral SRS imaging opens up a unique opportunity to shed new light on lipid metabolism."
Prof. Sunney Xie (Harvard University)
Fu, Dan; Yu, Yong; Folick, Andrew; Currie, Erin; Farese, Robert V., Jr.; Tsai, Tsung-Huang; Xie, X. Sunney; Wang, Meng C. "In Vivo Metabolic Fingerprinting of Neutral Lipids with Hyperspectral Stimulated Raman Scattering Microscopy," J. Am. Chem. Soc. 136, 8820-8828 (2014). link
Wang, Ping; Liu, Bin; Zhang, Delong; Belew, Micah Y., Tissenbaum, Heidi A.; Cheng, Ji-Xin "Imaging Lipid Metabolism in Live Caenorhabditis elegans Using Fingerprint Vibrations," Angew. Chem. Int. Ed. 2014, 53, 11787 –11792 (2014).
Wang, Meng C.; Min, Wei; Freudiger, Christian W.; Ruvkun, Gary; Xie, X. Sunney "RNAi screening for fat regulatory genes with SRS microscopy" Nat Methods 8, 135-138 (2011). link
Examples in Cancer Research
Researchers at Purdue have discovered a link between prostate cancer aggressiveness and cholesteryl esters which accumulate in lipid droplets inside cancer cells. This image shows liver metastasis of human prostate cancer, taken using a technique called stimulated Raman scattering microscopy. The bright dots represent intracellular lipid droplets enriched in cholesteryl ester.
Prof. Ji-Xin Cheng, Purdue University
Shuhua Yue, Junjie Li, Seung-Young Lee, Hyeon Jeong Lee, Tian Shao, Bing Song, Liang Cheng, Timothy A. Masterson, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng, "Cholesteryl Ester Accumulation Induced by PTEN Loss and PI3K/AKT Activation Underlies Human Prostate Cancer Aggressiveness," Cell Metabolism, 19, 3, 4 (2014) link
Therese S. Salameh, Thuc T. Le, Maxine B. Nichols, Erin Bauer, Ji-Xin Cheng, Ignacio G. Camarillo*, “An ex vivo Co-Culture Model System to Evaluate Stroma-Epithelial Interactions in Breast Cancer,” International Journal of Cancer, 132(2): 288-96 (2013). link
Examples in Bio-Othorgonal Imaging
"How to visualize the vast number of small biomolecules inside living cells represents a grand challenge, because these vital species cannot be labeled by bulky fluorophores. By labeling these target molecules with tiny vibrational tags such as alkyne moiety or stable isotopes and imaging the resulting chemicals by SRS microscopy, one could monitor metabolic dynamics of small bio-molecules in live cells and organisms with high sensitivity, specificity, resolution and bio-compatibility. Such an approach could do for small biomolecules what fluorescence imaging has done for larger species."
Prof. Wei Min (Columbia University)
L. Wei, Y. Yu, Y. Shen, W. C. Wang and W. Min. “Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy”, Proc. Natl. Acad. Sci. USA 110, 11226 (2013).
F. Hu, L. Wei, Y. Shen and W. Min. “Live-cell imaging of choline metabolites through stimulated Raman scattering coupled with isotope-based metabolic labeling”, Analyst, 139, 2312 (2014).
Y. Shen, F. Xu, L. Wei, F. Hu and W. Min. “Live-cell quantitative imaging of proteome degradation by stimulated Raman scattering”, Angew. Chem. Int. Ed. 53, 5596 (2014).
L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C. Lin, M. Wang and W. Min. “Live-cell imaging with alkyne-tagged small biomolecules by stimulated Raman Scattering”, Nature Methods, 11, 410 (2014).
Z. Chen, D. Paley, L. Wei, A. Weisman, R. Friesner, C. Nuckolls and W. Min. “Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette”, J. Am. Chem. Soc. 136, 8027 (2014).
L. Wei, Y. Shen, F. Xu, F. Hu, J. Harrington, K. Targoff and W. Min. “Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling”. ACS Chem. Biol. DOI: 10.1021/cb500787b (2015).