Probe design for super-multiplexed vibrational imaging.

Optical microscopy has served biomedical research for decades due to its high temporal and spatial resolutions. Among various optical imaging techniques, fluorescence imaging offers superb sensitivity down to single molecule level but its multiplexing capacity is limited by intrinsically broad bandwidth. To simultaneously capture a vast number of targets, the newly emerging vibrational microscopy technique draws increasing attention as vibration spectroscopy features narrow transition linewidth. Nonetheless, unlike fluorophores that have been studied for centuries, a systematic investigation on vibrational probes is underemphasized. Herein, we reviewed some of the recent developments of vibrational probes for multiplex imaging applications, particularly those serving stimulated Raman scattering (SRS) microscopy, which is one of the most promising vibrational imaging techniques. We wish to summarize the general guidelines for developing bioorthogonal vibrational probes with high sensitivity, chemical specificity and most importantly, tunability to fulfill super-multiplexed optical imaging. Future directions to significantly improve the performance are also discussed.