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.

Intravital microscopy is a uniquely powerful tool, providing the ability to characterize cell and organ physiology in the natural context of the intact, living animal. With the recent development of high-resolution microscopy techniques such as confocal and multiphoton microscopy, intravital microscopy can now characterize structures at subcellular resolution and capture events at sub-second temporal resolution. However, realizing the potential for high resolution requires remarkable stability in the tissue. Whereas the rigid structure of the skull facilitates high-resolution imaging of the brain, organs of the viscera are free to move with respiration and heartbeat, requiring additional apparatus for immobilization. In our experience, these methods are variably effective, so that many studies are compromised by residual motion artifacts. Here we demonstrate the use of IMART, a software tool for removing motion artifacts from intravital microscopy images collected in time series or in three dimensions.