Label‐Free Quantitative Imaging of Cholesterol in Intact Tissues by Hyperspectral Stimulated Raman Scattering Microscopy

Abstract

Current molecular analysis of cells and tissues routinely relies on separation, enrichment, and subsequent measurements by various assays. Here we demonstrate a platform of hyperspectral stimulated Raman scattering microscopy for fast, quantitative and label-free imaging of biomolecules in intact tissues using spectroscopic fingerprints as the contrast mechanism.

Live-cell bioorthogonal Raman imaging

“…Without labeling, SRS imaging cannot differentiate a specific type of lipids. To overcome this limitations, methods including tailored excitation [32] and hyperspectral SRS [33] have been developed for specifically imaging certain lipids with unique spectral features. In this regard, the bioorthogonal Raman imaging possesses the capability of easily differentiating lipid types even with similar chemical structures, by metabolic labeling with a Raman reporter, as demonstrated by SRS imaging palmitic acids in live cells using v-alkynyl palmitic acid (Alk-16) [22 ,23 ].…”

Biological imaging with coherent Raman scattering microscopy: a tutorial

“…78,80 In addition, the number of single-frequency CRS studies that use fingerprint contrast for biological imaging is steadily growing. 40,64 Although many molecular compounds in biology exhibit similar chemical groups, there are many others that can be uniquely identified by means of their vibrational signatures. Many exogenous molecules, such as pharmaceuticals and their carrier materials, have spectral features that are notably different from the spectral properties of tissues.…”

“…32 Yet, in other biological imaging applications, the separation of multiple spectral signatures is essential for identifying target compounds. 63,64 Improving the analytical capabilities of the CRS microscope is a subtle balancing act between fast image acquisition and expanding the instrument's sensitivity along the spectral dimension. Some of the many possible methods are sketched in Fig.…”

Label-free real-time imaging of myelination in theXenopus laevistadpole byin vivostimulated Raman scattering microscopy

“…Therefore, SRS microscopy enables us to probe molecule's chemical bond in cells or tissues and provides us with information regarding their molecular composition. [32][33][34][35] As illustrated in Fig. 1(a), the pump field (ω p ) and the Stokes field (ω S ) excite the molecules from the ground state to the excited vibrational state by passing through a virtual state.…”