Abstract 348: Label-Free High-Resolution Raman Imaging Reveals Distribution of Living Myocardium and Collagen in Rat Infarcted Heart
Label-free imaging of living myocardium and collagen in the infarcted heart has not been possible. Raman spectroscopy is now a confirmed optical tool allowing the analysis of molecular structure without labeling. Here, we for the first time applied a high-speed confocal laser Raman microscope, which we have recently developed to obtain images at faster speed, for molecular tissue imaging of rat myocardial infarct (MI) regions to elucidate whether cardiomyocytes and collagen can be identified. Experiments were performed on cardiac tissues excised from female Wistar rats at 28 days after the induction of MI by coronary ligation. Raman spectra were obtained from the cytosol of cardiomyocytes and fibrotic scar tissues by examining both freshly prepared and frozen-thawed specimens with RAMAN-11 (Nanophoton, Japan) with confirmation of the border-zone of MI using serial hematoxylineosin- and Masson’s trichrome-stained images. The samples were illuminated with a Nd:YAG laser (532 nm) through a water-immersion objective lens (× 60), and the Raman scattered light was guided to the slit (100 μm) of a spectrograph. The spectra were recorded by an electrically cooled CCD camera (−70°C, 15 bits, 1340 × 400 pixels). The following results were obtained from 10 hearts: Raman spectra of the cytosol of cardiomyocytes included the resonance Raman bands at 751, 1130, 1313, and 1582 cm−1 wavelengths arising from cytochrome c; Raman spectra of fibrotic tissues were well correlated with that of collagen type I; using principal component analysis, cardiomyocytes and collagen could be clearly imaged at the cellular level; and there were no significant differences in the positions of the Raman bands between the freshly prepared and frozen-thawed specimens. We note that Raman tissue imaging is capable of identifying both living myocardium and collagen with high resolution, owing to the utilization of the resonance Raman scattering of cytochrome c to identify the cardiomyocytes. This label-free molecular imaging method can be applied for real-time monitoring of the distribution of living myocardium and collagen in the human heart for the assessment of myocardial viability.