ABSTRACT
BACKGROUND: The use of conventional fluorescence microscopy to image biological systems at the cellular level is limited by its inability to spatially resolve thick tissues. We have applied the technique of multi-photon fluorescence microscopy to study the structure and function of endothelial cells in living human saphenous vein taken from patients undergoing coronary artery bypass surgery. MATERIALS AND METHODS: Vein segments were preserved for 1-4 h to determine the temporal effects of storage. The effect of pH on endothelial and smooth muscle cell viability was examined by storing segments at pH 6.0, 7.4, and 8.0. Calcein-mediated green fluorescence and ethidium homodimer-mediated red fluorescence were used to differentiate cell viability. Increases in diaminofluorescein fluorescence were used to measure bradykinin activation of endothelial nitric oxide synthase (eNOS) with or without N-nitro-l-arginine (L-NNA). Multi-photon imaging was performed with the BioRad MRC1024ES system. RESULTS: Successful imaging of endothelial and smooth muscle cells of vein segments was achieved. Cell viability was well preserved up to 3 h of storage but dramatically decreased after 4 h. Cell viability was maintained at pH 7.4, diminished at pH 8.0, and was completely lost at pH 6.0. A two- to threefold increase in eNOS activity was observed upon activation by bradykinin which was completely inhibited in L-NNA-treated samples. CONCLUSIONS: We have demonstrated the successful application of multi-photon microscopy in imaging and quantifying nitric oxide production and cell viability under various storage conditions in human saphenous veins. This imaging technique allows for the functional imaging of cellular processes and may have diagnostic potential in cardiovascular surgery for patients undergoing bypass operations.
