# Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching
## 3 Results and Discussion
Simultaneously captured multidepth multiphoton images of mouse pulmonary alveoli are shown in Figure 4. The mouse was sacrificed by using cervical dislocation. Its lung lobes were removed immediately and kept in chilled phosphate-buffered saline until imaging. Animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei University of Technology. Figure 4: Simultaneously acquired multiphoton images of mouse pulmonary alveoli at different depths, 2 μs/pixel, 512 × 512 pixels. (**a**) A pseudo-colored two-photon fluorescence (TPF) and second-harmonic generation (SHG) image acquired at the depth of 30 μm into the pulmonary parenchyma. Cyan pixels (obtained from CH1) are mainly derived from prominent SHG of collagen. The green arrow indicates a pronounced bundle of collagen fibers. Yellow pixels (obtained from CH2) are mainly derived from autofluorescence of elastin, macrophages, and surfactant proteins. The yellow arrow denotes notable elastin fibers. The white arrow indicates surfactant proteins gathered probably in the cytoplasm of a type II pneumocyte. The blue arrow shows that collagen fibers spiral around elastin fibers. (**b**) A pseudo-colored TPF and SHG image simultaneously acquired at the depth of 35 μm into the pulmonary parenchyma. The red arrows in (**a,b**) denote the sections of the same macrophages at both depths. The scale bar is 40 μm. The wavelength of the mode-locked Ti:Sapphire laser was kept at 870 nm. The detection channel CH1 was turned on to collect SHG around 435 nm, and CH2 was applied to detect autofluorescence in the range of 495-635 nm. At the focal planes, both ARM1 and ARM2 have average powers of approximately 10 mW. The images were acquired at the depths of 30 (ARM1) and 35 (ARM2) into the pulmonary parenchyma. Cyan and yellow pixels in the images were captured from CH1 and CH2, respectively. As shown in Figure 4a, lumens of alveoli filled with air are apparent, and the cyan pixels may be predominantly contributed by prominent SHG from collagen fibers in the alveolar extracellular matrix, according to their typical bundle-like structures. The green arrow indicates a notable bundle of collagen fibers. The yellow pixels denoted by the yellow arrow are attributed to be elastin fibers, according to their typical rope-like structures. The blue arrow depicts that collagen bundles spiral around elastin fibers to form a condensed composite structure. This structural feature confirms that elastin fibers and collagen bundles are actually separate structures, though their signals seem to overlap in some pixels. The round-shaped yellow objects indicated by the red arrows may correspond to macrophages. The size of a macrophage is approximately 10-15 (m). The endogenous contrasts of elastin fibers and macrophages are predominantly contributed by autofluorescence emission at 495 to 635 nm, which is consistent with previously published data [31]. Gathered granules denoted by the white arrow are most likely derived from autofluorescence of surfactant proteins along luminal surfaces (probably inside the cytoplasm of type II pneumocytes). Scattered granules around collagen and elastin fibers are probably contributed by autofluorescence derived from lipopigments and flavins in fibroblasts [32]. The experimental results confirm that the imaging system is capable of revealing the microenvironment of mouse pulmonary alveoli. Figure 4b is an image obtained at the depth of 35 (m), which has the same biological components as Figure 4a. The white circles in Figure 4a,b outline the same alveolus, indicating that the focal planes switch in the axial direction without tilt. Note that images obtained at different depths have similar image qualities, proving that spherical aberrations introduced by objectives O1 and O2 are negligible. The red arrows in Figure 4b denote the same two macrophages indicated in Figure 4a, but they are imaged at different sections.