Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure

Background: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. Method: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. Results: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. Conclusion: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.