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 microm.


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 wide-field 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.
van Zandvoort
Marc
M
Department of Biophysics, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands. MAMJ.vanZandvoort@BF.unimaas.nl
Engels
Wim
W
Douma
Kim
K
Beckers
Linda
L
Oude Egbrink
Mirjam
M
Daemen
Mat
M
Slaaf
Dick W
DW
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D002340:Carotid Artery Diseases / Q000473:pathology*; D005782:Gels; D007962:Leukocytes; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D012685:Sepharose; D016610:Tissue Embedding
D001057:Apolipoproteins E; D005782:Gels; D012685:Sepharose