Tracking X-ray microscopy for alveolar dynamics in live intact mice

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1 SUPPLEMENTARY INFORMATION Tracking X-ray microscopy for alveolar dynamics in live intact mice Soeun Chang, Namseop Kwon, Byung Mook Weon, Jinkyung Kim, Chin Kook Rhee, Han Sung Choi, Yoshiki Kohmura, Masaki Yamamoto, Tetsuya Ishikawa, and Jung Ho Je Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Table 1 Supplementary Table 2 Supplementary Discussion 1 Supplementary Discussion 2 Overall lung movements Movement trajectories of individual alveoli during respiration Alveolar size (diameter) change correlated with the ventilator pressure Alveolar pressure-volume curves in mice The pressure size curves of two additional alveoli in two different mice Morphologies of duct and sac alveoli Morphological changes of individual alveoli in 3-D geometry during respiration Alveolar morphology Diameters of alveoli Morphologies of individual duct alveoli (DA) and sac alveoli (SA) Ratio of alveolar depth (D) to mouth diameter (MD) Radiation dose Note: Supplementary Videos 1-8 are available on the Scientific Reports website

2 Video Legends Supplementary Video 1. Real-time movie of the upper right lung lobe in a mouse during respiration with a normal tidal volume (160µl) (Supplementary Fig. 1). Here the overall lung movement during respiration is significant as more or less 300μm even for the upper right lobe with a minimum lung movement. Supplementary Video 2. Microtomographic movie of the upper right lung lobe of a mouse, taken at every end point of inspiration during 180º rotation of the sample stage (Supplementary Fig. 6a). Supplementary Video 3. Microtomographic movie of the upper right lung lobe of a mouse, taken at every end point of expiration during 180º rotation of the sample stage (Supplementary Fig. 6b). Supplementary Video 4. Real-time movie of individual alveoli from the end point of expiration to the end point of inspiration in the upper right lung lobe of a mouse (Supplementary Fig. 1a,b,c). Different movement trajectories of individual alveoli enabled us to recognize the coherent motion of optical features of each alveolus during respiration and as a result to identify and track individual alveoli from the overlapped images of numerous alveoli

3 Supplementary Video 5. Segmented microtomographic movie of one unit of branched ducts and sacs with alveoli in inspiration (Fig. 3a). Supplementary Video 6. Segmented microtomographic movie of one unit of branched ducts and sacs with alveoli in expiration (Fig. 3a). Supplementary Video 7. Segmented microtomographic movie of the upper right lung lobe of a mouse in inspiration (Supplementary Fig. 6c). Each color indicates each unit of branched ducts and sacs with alveoli. Supplementary Video 8. Segmented microtomographic movie of the upper right lung lobe of a mouse in expiration (Supplementary Fig. 6d). Each color indicates each unit of branched ducts and sacs with alveoli

4 Supplementary Figure 1 Representative real-time microradiographs of the upper right lung lobe of a mouse during a normal inspiration (0~200ms)/expiration (200~600ms) cycle with a normal tidal volume (160µl). The arrow indicates the alveolus at the apex top of the upper right lobe. Here the overall lung movement during respiration is significant as ~ 300 m even for the upper right lobe with a minimum lung movement. Scale bar, 100µm

5 Supplementary Figure 2 (a-c) Representative real-time microradiographs of the upper right lung lobe of a mouse from the end point of expiration (t 1 ) to the end point of inspiration (t 3 ). Different movement trajectories of individual alveoli enabled us to recognize the coherent motion of optical features of each alveolus during respiration and as a result to identify and track individual alveoli from the overlapped images of numerous alveoli. Each color (blue, green, red, and yellow) of the dashed circles indicates the same alveolus. (d) Trajectories (dashed arrows) of individual alveoli movements during respiration. Scale bar, 50µm. (e) The trajectories of 60 alveoli in 6 different mice during inspiration and expiration. The trajectories, defined here as the distance of individual alveolar positions in end of expiration and end of inspiration

6 Supplementary Figure 3 Alveolar size (diameter) change correlated with the ventilator pressure. (a) The ventilator pressure with time. (b) The size change of an alveolus with time (8ms interval) by real-time microradiography. Blinded analyses of the same alveolar images were done by five independent investigators, showing an inter-observer coefficient of variation of alveolar size measurements of <3%

7 Supplementary Figure 4 Alveolar pressure-volume curves in mice. Our data (red circles) clearly show a closed-loop hysteresis, similar to the whole-lung curve (inset) 1. We note that alveoli in live intact mice show lower compliances than reported for alveoli of thorax-incised mice (grey circles) 2. Here normalized volumes were estimated based on using alveolar size (our data of Fig. 2c) and alveolar area (IVM)

8 Supplementary Figure 5 The pressure size curves of four additional alveoli in four different mice during inspiration and expiration in the tidal volume ventilation. The alveolar sizes were normalized to the sizes measured at the end of expiration. The four alveoli show similar hysteresis behaviors. The size measurements were performed over 2 respiration cycles in each case

9 Supplementary Figure 6 Morphologies of duct and sac alveoli. (a-b) The microradiographs of the upper right lung lobe of a mouse in inspiration (a) and expiration (b). (c-d) The segmented 3D images corresponding to the images in (a) and (b), respectively. Each color indicates each unit of branched ducts with duct and sac alveoli. The diameter increments of individual alveolar ducts in respiration, estimated from the 3-D images in (c) and (d), are small as 4.3±1.3 % (mean ± s.e.m.). Scale bars, 100µm

10 Supplementary Figure 7 Morphological changes of individual alveoli in 3-D geometry during respiration. The ratio of the alveolar depth (D) and the mouth diameter (MD), D/MD; D/MD in sac alveoli (SA): 0.87±0.03 (mean±s.e.m.) in expiration (light gray) and 0.86±0.03(mean±s.e.m.) in inspiration (dark gray; P=0.8), D/MD in duct alveoli (DA): 0.59±0.03 (mean±s.e.m.) in expiration (light gray) and 0.59±0.03 (mean±s.e.m.) in inspiration (dark gray; P=1.0); *P=1.1x10-7 (**P=1.3x10-7 ) for D/MD between SA and DA in expiration (inspiration). n = ~80 alveoli over 18 ducts with sacs in a live intact mouse

11 Supplementary Figure 8 Alveolar morphology. (a-b) Alveolar schematics of hyper-hemisphere and hypo-hemisphere. (c-d) Representative 3D segmented images of alveolar epithelial surfaces for a sac alveolus (c) and a duct alveolus (d). The local distortion or flats on the surface in the 3-D segmented images are presumably due to adjacent alveoli and diversity in duct branch directions

12 Supplementary Table 1 Raw data of Fig. 2b. d 1 (d 2 ): diameter of individual alveoli in expiration (inspiration), I: diameter increment, and M: mouse number

13 Supplementary Table 2 Morphologies of individual duct alveoli (DA) and sac alveoli (SA) in 3-D geometry during respiration, measured for ~ 80 alveoli over 18 ducts with sacs in a live intact mouse (the errors correspond to the s.e.m.)

14 Supplementary Discussion 1. Ratio of Alveolar Depth (D) to Mouth Diameter (MD). The ratio D/MD is critically associated with the flow patterns within alveoli. One aspect of this factor can be thus exemplified by the study on flow trajectories of particles to predict deposition sites of inhaled pharmaceutical aerosols or pollutants in alveoli 3,4. Despite its importance, the reported D/MDs show a large variation from 0.4 to 2.4 3,4, mostly by the absence of real-time study for 3D alveoli imaging in live intact mice. The D/MD values (0.59±0.02 in ducts and 0.87±0.02 in sacs), obtained for live intact mice in this study, may greatly contribute to the understanding of flow patterns in alveoli 3,4. Supplementary Discussion 2. Radiation Dose. The high coherence of the monochromatic X-rays permitted to observe alveolar boundaries of live mice with high visibility by the edge-refraction enhancement 5 8. Actually the edge enhancement is very useful to reduce the radiation dose. In this study, the total radiation dose for one set (inspiratory triggering + expiratory triggering) of microtomography was reasonably small as ~ 2.5Gy 7, in spite of very high resolution 3-D imaging

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