Direct Counting and Imaging Chain Lengths of Lipids by Stimulated Raman Scattering Microscopy

Direct counting and mapping the chain lengths of fatty acids on a microscopic scale are of particular importance but remain an unsolvable challenge. Although the current hyperspectral stimulated Raman scattering (SRS) microscopy has gained exceptional capability in chemical imaging of the degree of desaturation, the complete lipid characterization, including the carbon chain length quantification, is awaiting a major breakthrough. Here, we pushed the spectral resolution limit of hyperspectral SRS microscopy to 5.4 cm^-1 by employing a highly efficient spectral compressor, which achieved spectral narrowing of the fs laser without much energy loss. The SRS imaging with such high spectral resolution enabled us to differ eight types of saturated lipids with carbon chain lengths from C8:0 to C22:0 by interrogating their subtly red-shifting Raman bands of alkyl C-C gauche stretches between 1070 and 1110 cm^-1. The SRS microscopy with superior spectral resolution will pave the way for comprehensive lipid characterization and contribute to uncovering the abnormal pathways of lipid metabolism in cancer.