Two-Photon Imaging within the Murine Thorax without Respiratory and Cardiac Motion Artifact

Abstract

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 of the lung: minimizing invasiveness

“…The animal preparation prior to imaging is performed according to this consensual protocol [11,18,[23][24][25][26][27][28][29]:…”

“…The use of this strategy allows investigation of vascular and circulatory disorders [25,[36][37][38], study of the microvascular endothelial activation in response to oxidative stress inducers (Figure 2c) [28], measurement of the pulmonary endothelial surface layer of mice [35,39], and the phagocytic capacity of pulmonary immune cells [40][41][42].…”

“…The lung is maintained by suction with a force great enough to lessen the physiological motion into a level compatible with high-resolution dynamic microscopic imaging [18,24,28,29,[43][44][45][46][47][48][49][50]. In particular, the resolution reached using this technique recently permitted observation of dendritic cell dendrites (Figure 2d) [44] and patrolling monocytes controlling tumor metastasis to the lung [51].…”

“…To a certain extent, the use of an inverted microscope might help microscopic imaging by facilitating the contact between the objective and lung, generally through the coverslip on which the animal lays [23,24,28,35,45]. However, perturbations coming from breathing remain [45] and thus requires the combined use of the inverted microscope with another stabilization technique.…”

“…Adapted to lung IVM, this technique is generally used in association with other stabilization techniques [23,25,28,45,56]. To our knowledge, this technique has never been used for free-moving lung IVM of mice without slowing the respiratory rate (see section 'Oversampling of free-moving lung') because of the hardly-reproducible motion coming from the heartbeat-breathing combination.…”