Spontaneous Raman imaging has emerged as powerful label-free technique for investigating the molecular composition of medicines and biological specimens. Although Raman imaging can facilitate understanding of complex biological phenomena in vivo, current imaging modalities are limited in speed and sample compatibility. Here, we introduce a single-objective line-scanning light-sheet microscope, named
-OPM, which records Raman images on a timescale of minutes to seconds. To demonstrate its function, we use
-OPM to map and identify microplastic particles based on their Raman spectral characteristics. In live zebrafish embryos, we show that
-OPM can capture wound dynamics at five-minute intervals, revealing rapid changes in cellular and extracellular matrix composition in the wounded region. Finally, we use
-OPM to synchronize and average 36,800 individual frames to obtain hyperspectral videos of a zebrafish embryo’s beating heart at an effective 28 frames per second, recording compositional changes throughout the cardiac cycle.