Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy

Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.