Longitudinal live-cell chemical imaging using coherent Raman scattering microscopy.

Optical methods enable minimally invasive analysis of chemical distributions and dynamics in biological specimens. Many optical imaging modalities have been developed to generate chemical contrast based on intrinsic molecular signatures or highly specific labeling. Coherent Raman scattering (CRS) microscopy has emerged as a powerful label-free technique for imaging biomolecules or exogenous compounds in biological samples. Most CRS imaging studies have focused on extracting chemical distribution, composition, and abundance in samples, and work in these areas has been extensively reviewed. However, an important aspect of chemical processes-the dynamics of chemical changes over time-has received less emphasis in CRS microscopy. Many critical insights into chemical processes lie in the spatiotemporal evolution of molecular species in live samples. Such information can only be obtained through live-cell imaging, and more importantly, longitudinal imaging of the same sample over time. In this review, we focus on key parameters related to longitudinal live-cell CRS microscopy and major recent studies. We first discuss three primary factors that must be considered for longitudinal CRS imaging: limit of detection, chemical selectivity, and phototoxicity. Limit of detection and chemical selectivity determine what CRS can measure, whereas phototoxicity dictates whether longitudinal imaging can capture meaningful cellular chemical dynamics with minimal perturbation to biological functions. In these discussions, we also provide critical comparisons between CRS microscopy and fluorescence microscopy. We then review recent developments and achievements in longitudinal CRS microscopy. Finally, we provide perspectives on future directions and areas where longitudinal CRS microscopy can make contributions to advancing our understanding of biological processes.