Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study

Background: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the ( sub) cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study ( sub) cellular details in arteries with wall thicknesses above 50 mum. Methods: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. Results: In thin slices, ( sub) cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E-/- mice demonstrated that in contrast to confocal or widefield microscopy, TPLSM can be used to visualize ( sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E-/- mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. Conclusions: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse. Copyright (C) 2004 S. Karger AG, Basel.