van Zandvoort Marc, Engels Wim, Douma Kim, Beckers Linda, Oude Egbrink Mirjam, Daemen Mat, Slaaf Dick W
Department of Biophysics, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands.
J Vasc Res. 2004 Jan-Feb;41(1):54-63. doi: 10.1159/000076246. Epub 2004 Jan 16.
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.
Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries.
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.
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.
在完整动脉内（亚）细胞水平上同时进行结构和功能信息的离体和在体成像，将极大地有助于了解动脉粥样硬化的发生机制。由于穿透深度有限以及分辨率随深度降低，活体和共聚焦荧光显微镜不适用于研究壁厚超过50微米的动脉中的（亚）细胞细节。
我们使用结合了三维分辨率和大穿透深度的双光子激光扫描显微镜（TPLSM）对小鼠颈动脉进行成像。
在薄片中，使用组织化学技术识别的（亚）细胞结构也可以通过TPLSM识别。在离体条件下，对Apo-E(-/-)小鼠完整动脉粥样硬化动脉进行的结构实验表明，与共聚焦或宽视野显微镜不同，TPLSM可用于可视化动脉粥样硬化斑块的（亚）细胞结构细节。在体实验中，我们对野生型C57BL6小鼠的健康动脉和Apo-E(-/-)小鼠的动脉粥样硬化动脉进行了初步实验。作为功能测量的一个例子，我们在体观察到了管腔内荧光标记的白细胞。此外，局部染色后获得了血管壁和动脉粥样硬化斑块的详细形态学信息。
因此，TPLSM有可能实现功能和结构的联合研究，因此非常适合研究小鼠动脉粥样硬化发生过程中细胞水平的结构-功能关系。
