Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy


Label-free microscopy that has chemical contrast and high acquisition speeds up to video rates has recently been made possible using stimulated Raman scattering (SRS) microscopy. SRS imaging offers high sensitivity, but the spectral specificity of the original narrowband implementation is limited, making it difficult to distinguish chemical species with overlapping Raman bands. Here, we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species, based on tailored multiplex excitation of its vibrational spectrum. This is implemented by spectral modulation of a broadband pump beam at a high frequency (>1 MHz), allowing detection of the SRS signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live Caenorhabditis elegans.

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Figure 1: Principle of SRS.
Figure 2: Excitation schemes of SRS.
Figure 3: Spectral modulation scheme.
Figure 4: STE-SRS microscopy setup.
Figure 5: Characterization of STE-SRS.
Figure 6: Imaging of lipid storage in C. elegans.


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The authors thank Linjiao Luo and Aravinthan Samuel for providing the C. elegans sample for initial testing, B. Saar and Sijia Lu for helpful discussions and comments on the manuscript, and Xu Zhang for assisting in the final concentration measurements. C.W.F. acknowledges Boehringer Ingelheim Fonds for a PhD Fellowship. This work was supported by the National Institutes of Health (NIH) Director's Pioneer Award and NIH TR01 grant 1R01EB010244-01.

Author information

C.W.F., W.M. and X.S.X. conceived the idea and drafted the manuscript. C.W.F. and G.R.H. built the instrument, B.X. and M.D. designed and built the pulse-shaper, and C.W.F. conducted the experiments.

Correspondence to X. Sunney Xie.

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Freudiger, C., Min, W., Holtom, G. et al. Highly specific label-free molecular imaging with spectrally tailored excitation-stimulated Raman scattering (STE-SRS) microscopy. Nature Photon 5, 103–109 (2011). https://doi.org/10.1038/nphoton.2010.294

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