# Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching
## 3 Results and Discussion  
The capability of the real-time multidepth multiphoton microscopy was demonstrated by using freshly excised mouse lung tissues. As shown in Figure 3, the lower respiratory tract, along the proximal-distal axis, includes extrapulmonary airways (i.e., trachea and primary bronchi), intrapulmonary airways (i.e., lobar bronchi, segmental bronchi, subsegmental bronchi, and bronchioles), and alveolar regions. Figure 3: Schematic of mouse lower respiratory tract. The lower respiratory tract, along the proximal-distal axis, includes extrapulmonary airways, intrapulmonary airways, and alveolar regions. The alveolar regions are composed of several distinct layers, i.e., pneumocytes, epithelial basement membrane, intersititium, endothelial basement membrane, and endothelial cells. The pneumocytes are composed of type I pneumocytes responsible for air exchange and type II pneumocytes secreting surfactant proteins. A few macrophages may be present in the alveolar lumens or intersititium. Fibroblast can be found in the intersititium. Figure 2: Demonstration of real-time multidepth multiphoton imaging using pixel-to-pixel focus-switching, 2 μs/pixel, 200 × 200 pixels for (**c**), 100 × 100 pixels for panels (**d**,**e**). (**a**) The pixel clock from the microscope. (**b**) Pulse train from the function generator. (**c**) Autofluorescence image of proflavine in aqueous solution. Signals obtained from the depths of 0 and 5 μm are displayed in different columns of the same image. The inset is a zoomed-in view of 2 × 4 pixels. (**d**,**e**) are autofluorescence images extracted from different columns of (**c**), corresponding to the depths of 0 and 5 μm, respectively. The scale bars are 10 μm. At the distal end of the lower respiratory tract lie the alveolar regions. We first describe the structure of mouse alveolar regions and available multiphoton imaging modalities. Alveolar regions are composed of alveoli and a network of capillaries. Alveoli are small air sacs inside lungs, allowing air exchange. Along the luminal surface, an alveolus is lined by a cell layer of squamous type I pneumocytes and cuboidal type II pneumocytes. Type I pneumocytes provide an extensive surface area for air exchange, and type II pneumocytes secrete pulmonary surfactant proteins. The pneumocytes are anchored to the subjacent epithelial basement membrane, which is apposed and often fused to the endothelial basement membrane of a surrounding capillary. Endothelial cells of the capillary adhere closely to the endothelial basement membrane. There is interstitium between the two basement membranes, where a few stromal cells (e.g., fibroblasts) may be present [28]. The epithelial basement membrane, interstitium, and endothelial basement membrane compose the extracellular matrix of an alveolus. Two main components of the extracellular matrix are collagen and elastin fibers, which are responsible for the tensile strength and elastic modulus of the alveoli, respectively. Collagen and elastin fibers can be revealed by contrast from SHG and autofluorescence, respectively. Fibroblasts in the interstitium keep synthesizing ground substance, as well as secreting collagen and elastin fibers, to maintain the integrity of the extracellular matrix. Pulmonary diseases, e.g., hyperplasia, fibrosis, and neoplasm, will cause prominent structural damage to the alveolar extracellular matrix. Besides the main structures, a few macrophages also reside in the alveoli, which can be imaged by autofluorescence predominantly from lipids and flavins [29]. Accumulation and migration of macrophages may indicate inflammation or asthma. In conclusion, researchers can use multiphoton imaging to study the morphologies of mouse alveoli for identifying structure-associated diseases and to observe cellular dynamics for monitoring physiological processes. Furthermore, because of their similar structures, mouse alveoli are good models for further investigation of human alveoli [28; 30].