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

2011; Nature Portfolio; Volume: 5; Issue: 2 Linguagem: Inglês

10.1038/nphoton.2010.294

1749-4893

Christian W. Freudiger, Wei Min, Gary R. Holtom, Bingwei Xu, Marcos Dantus, X. Sunney Xie,

Advanced Fluorescence Microscopy Techniques

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. Spectral modulation of a broadband pump beam allows sensitive and specific molecular imaging based on stimulated Raman scattering. Measurements of cholesterol, protein, and stearic and oleic acid are reported.