Instrumentation and applications of hyperspectral stimulated Raman scattering microscopy
Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography-mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real- time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system. With high imaging speed and 3D spatial resolution, coherent Raman scattering (CRS) microscopy is enabling a new approach for real- time vibrational imaging of single cells in a living system. Stimulated Raman scattering (SRS) rises among the CRS processes due to its many figures of merits. The detected intensity change because of a Raman transition is proportional to Im[χ (3)]IpIS, where χ(3) represents the third-order nonlinear susceptibility, and I refers to the intensity of each field. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10,000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.
Cheng, Purdue University.
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