Light microscopic imaging of blood vessels and distribution of serum proteins is essential to analyze hemodynamics in living animal lungs under normal respiration or respiratory diseases. In this study, to demonstrate dynamically changing morphology and immunohistochemical images of their living states, "in vivo cryotechnique" (IVCT) combined with freeze-substitution fixation was applied to anesthetized mouse lungs. By hematoxylin-eosin staining, morphological features, such as shapes of alveolar septum and sizes of alveolar lumen, reflected their respiratory conditions in vivo, and alveolar capillaries were filled with variously shaped erythrocytes. Albumin was usually immunolocalized in the capillaries, which was confirmed by double-immunostaining for aquaporin-1 of endothelium. To capture accurate time-courses of blood flow in peripheral pulmonary alveoli, glutathione-coated quantum dots (QDs) were injected into right ventricles, and then IVCT was performed at different time-points after the QD injection. QDs were localized in most arterioles and some alveolar capillaries at 1 s, and later in venules at 2 s, reflecting a typical blood flow direction in vivo. Three-dimensional QD images of microvascular networks were reconstructed by confocal laser scanning microscopy. It was also applied to lungs of acute pulmonary hypertension mouse model. Erythrocytes were crammed in blood vessels, and some serum components leaked into alveolar lumens, as confirmed by mouse albumin immunostaining. Some separated collagen fibers and connecting elastic fibers were still detected in edematous tunica adventitia near terminal bronchioles. Thus, IVCT combined with histochemical approaches enabled us to capture native images of dynamically changing structures and microvascular hemodynamics of living mouse lungs.
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