Multiplexed Raman microscopy

A new version of stimulated Raman scattering (SRS) microscopy speeds up image acquisition by allowing multiple bands to be measured simultaneously (J. Am. Chem. Soc., DOI: 10.1021/ja210081h). Dan Fu, Xiaoliang Sunney Xie, and coworkers at Harvard University and the University of Notre Dame achieve the multiplexing by adding an acousto-optic tunable filter (AOTF) to their SRS setup. In the AOTF, acoustic waves sent through a crystal diffract the broadband femtosecond pump laser beam into a number of bands, each corresponding to a Raman shift modulated at a different frequency. The filtered pump beam is combined with a picosecond laser beam and sent to a laser scanning microscope. The Raman shift is determined from the energy difference between the filtered pump beam and Stokes beam, which is encoded in the modulation frequency. The researchers use a Fourier transform to extract multiple Raman bands by demodulating the signal at the different frequencies. Using the method, the researchers measured the biochemical composition of microalgae. They used three channels to distinguish among pigments, lipids, and proteins in the cells. They also obtained images of blood, lipids, and proteins in freshly excised mouse ear tissue.