Center for Immunobiology, Indiana University, Indianapolis, Indiana, USA.
Am J Pathol. 2011 Jul;179(1):75-82. doi: 10.1016/j.ajpath.2011.03.048. Epub 2011 May 7.
Intravital microscopy has been recognized for its ability to make physiological measurements at cellular and subcellular levels while maintaining the complex natural microenvironment. Two-photon microscopy (TPM), using longer wavelengths than single-photon excitation, has extended intravital imaging deeper into tissues, with minimal phototoxicity. However, due to a relatively slow acquisition rate, TPM is especially sensitive to motion artifact, which presents a challenge when imaging tissues subject to respiratory and cardiac movement. Thoracoabdominal organs that cannot be exteriorized or immobilized during TPM have generally required the use of isolated, pump-perfused preparations. However, this approach entails significant alteration of normal physiology, such as a lack of neural inputs, increased vascular resistance, and leukocyte activation. We adapted techniques of intravital microscopy that permitted TPM of organs maintained within the thoracoabdominal cavity of living, breathing rats or mice. We obtained extended intravital TPM imaging of the intact lung, arguably the organ most susceptible to both respiratory and cardiac motion. Intravital TPM detected the development of lung microvascular endothelial activation manifested as increased leukocyte adhesion and plasma extravasation in response to oxidative stress inducers PMA or soluble cigarette smoke extract. The pulmonary microvasculature and alveoli in the intact animal were imaged with comparable detail and fidelity to those in pump-perfused animals, opening the possibility for TPM of other thoracoabdominal organs under physiological and pathophysiological conditions.
活体显微镜因其能够在保持复杂自然微环境的情况下对细胞和亚细胞水平进行生理测量而得到认可。双光子显微镜 (TPM) 使用比单光子激发更长的波长，从而将活体成像扩展到更深的组织，同时光毒性最小。然而，由于采集速度相对较慢，TPM 特别容易受到运动伪影的影响，当对受呼吸和心脏运动影响的组织进行成像时，这是一个挑战。无法在 TPM 期间外展或固定的胸腹部器官通常需要使用隔离、泵灌注的制剂。然而，这种方法需要对正常生理进行重大改变，例如缺乏神经输入、血管阻力增加和白细胞激活。我们采用了活体显微镜技术，使 TPM 能够对活的、呼吸的大鼠或小鼠的胸腹腔内的器官进行成像。我们获得了完整肺的扩展活体 TPM 成像，肺可能是最容易受到呼吸和心脏运动影响的器官。活体 TPM 检测到肺微血管内皮细胞激活的发展，表现为白细胞黏附和血浆渗出增加，对 PMA 或可溶性香烟烟雾提取物等氧化应激诱导剂有反应。完整动物的肺微血管和肺泡的成像具有与泵灌注动物相当的细节和保真度，为在生理和病理生理条件下对其他胸腹部器官进行 TPM 成像开辟了可能性。
