Figures
- Fig. 1—
a) Diagram of the Cellvizio®-Lung System (Mauna Kea Technologies, Paris, France), and relative sizes of the 1.4-mm probe tip and the b) bronchiolar and c) alveolar structures. a) The fibred confocal fluorescence microscopy system is made of several components: a 1.4-mm miniaturised flexible fibred miniprobe made of thousands of fibre cores (upper right), a 488-nm excitation wavelength laser source, an XY scanning system (upper left), and a dedicated control and acquisition software that allows the reconstruction of dynamic images in real time. Note that the spectroscopy channel is not represented in the diagram; complete details of the spectroscopy system have been previously described 4. b) The tip of the miniprobe is superimposed on a distal bronchiole lumen in a 7-μm fixed lung tissue section. c) The miniprobe is superimposed in front of an alveolar duct system. The white bar corresponds to the diameter of the fibre bundle (600 μm).
- Fig. 2—
a–c) Fibred confocal fluorescence microscopy (FCFM) images of a well preserved lung sample fixed in glutaraldehyde at 20 cmH2O pressure, and d–f) comparison with corresponding 10-μm unstained histology sections, imaged using an oil immersion epifluorescence microscope. Both imaging modalities were performed at a 488-nm excitation wavelength. a) FCFM imaging of an alveolar duct system showing fine details of the capillary network on the alveolar walls (black arrow). Images were obtained after applying the probe onto the alveolated section of the sample. See online supplementary video for the dynamic sequences. b) FCFM image of the lung seen from the pleural surface. #: alveolar wall connecting to the peripheral connective tissue of the pleura. c) FCFM imaging from inside a bronchus showing an en face view of the epithelial layer. Goblet mucus cells appear as black spots (white arrow). d) Fluorescence microscopy of a 10-μm thick section corresponding to the alveolar wall of a), showing details of fixative-induced fluorescent erythrocytes in the alveolar capillary network (black arrow). e) Fluorescence microscopy, pleural section and adjacent alveolar walls. f) Fluorescence microscopy of the bronchial section corresponding to (c). White arrow: goblet cells with cytoplasmic mucus appearing in black. Scale bars = a–c) 50 μm, d) 10 μm, e) 25 μm and f) 20 μm.
- Fig. 3—
In vivo alveolar confocal fluorescence imaging according to the relative positions of the probe tip and the alveolar duct axis (nonsmoker healthy volunteers). a–c) The different points of view of the alveolar duct are indicated according to d) schematic Weibel’s representation of the peripheral lung elastic backbone (modified from 8 with permission from the publisher). Thick coloured arrows: supposed angles of penetration of the miniprobe into the alveolar unit. In nonsmokers, fibred confocal fluorescence microscopy (FCFM) mainly imaged the elastin that is concentrated in the axial fibred system (represented as orange and yellow coloured lines in d)), which forms the edges of the alveolar duct rim region (yellow) and the alveolar ring entrances (orange). a) Oblique view of the duct axial fibred system, showing the helical or looped shape of the duct’s edge (arrows). b) Lateral view of the axial system, showing how the elastin fibres at the alveolar openings are interconnected with the frame of the alveolar duct, in a “ladder-stitch” manner. Two sequential images show the elongation of the alveolar duct along its longitudinal axis in synchrony with the respiration (online video 7). White bars: size variations of the alveolar entrances. c) View down the axis of the duct, showing alveolar ring entrances in the back planes (#), due to a slight compression effect (see online video 6 for a dynamic sequence). Scale bars = 50 μm.
- Fig. 4—
Alveolar mouth (a, c and d) and lobular microvessel (b, e and f) measurements. Distribution of a) alveolar mouth and b) lobular microvessel diameters. c and d) Examples of alveolar ring measurements. White arrows: size of the entrance ring at minimal compression. e and f) Examples of extra-alveolar microvessel measurements. Black arrows: diameters measured upstream of the vessel bifurcation. Scale bars = 50 μm.
- Fig. 5—
a) Ex vivo and b) in vivo consequences of a probe tip compression applied on the lung tissue in the axis of the alveolar duct. From left to right: effect of increasing pressure on the tissue (see online video for the dynamic sequence). a) Ex vivo fixed sample. b) In vivo fibred confocal fluorescence microscopy images from a nonsmoking individual. Dashed arrows: edges of the alveolar duct; arrows: successive appearance of distant alveolar duct structures. Compression allowed for visualisation of deeper structures down the axis of the duct, providing a 3D-like effect, without perceptible deformation in the first plane of focus. Scale bars = 50 μm.
- Fig. 6—
In vivo fibred confocal fluorescence microscopy alveolar imaging in smokers. a) Alveolar system filled with highly fluorescent alveolar macrophages. Axial view down the alveolar duct. The corresponding image in a nonsmoker can be found in figure 3c⇑. Dynamic imaging can be seen in online video 9. b) Alveolar duct, oblique view. Arrows: helical arrangement of the axial cables of the alveolar duct. The corresponding image in a nonsmoker can be found in figure 3a⇑. c) In vivo imaging of the alveolar walls, showing bubbles on the surface of the alveolar wall and alveolar entrance (dashed arrows; see online video). d) Fine details of the alveolar wall from the same patient as in a). Lower left: fluorescent alveolar macrophages; upper right: triple lines of alveolar wall at the junction of three alveoli; middle: linear structures that may correspond to pleats of alveolar wall or to the relief of the capillary within the alveolar space. Dynamic imaging can be visualised at the end of the online video sequence. Scale bars = 50 μm.
- Fig. 7—
Autofluorescence emission spectra of alveolar systems from a) nonsmokers and b) healthy smokers, during in vivo alveoscopy. Blue: elastin powder; red: nonsmoker alveoli; green, pink, yellow: smoker alveoli; black: smoked cigarette. AU: arbitrary units.
- Fig. 8—
Fibred confocal fluorescence microscopy (FCFM) cellular imaging in smoking individuals. a) In vivo FCFM imaging of macrophage alveolitis. Correlation between the number of cigarettes smoked per day and the number of fluorescent macrophages observed per field of view (r = 0.70, p<0.0001; Spearman’s correlation test). b) Alveolus/bronchus fluorescence intensity ratio. Alveolar fluorescence intensity appeared linked to the intensity of the macrophage alveolitis (r = 0.50, p<0.0001; Spearman’s correlation test). c) Large activated macrophages in smokers. Correlation between the number of cigarettes smoked per day and the mean number of macrophages >30 μm per explored alveolar area (r = 0.77, p = 0.0002; Spearman’s correlation test). d) In vivo FCFM imaging of macrophage mobility. The number of cigarettes smoked per day appeared higher in subjects presenting mobile alveolar macrophages (MM; mean±sd 16.5±4.35 cigarettes·day−1) compared with those with immobile macrophages (IM; 6.75±4.08 cigarettes·day−1; p = 0.0019; Mann–Whitney test). Data are presented as mean±sd.
Tables
- Table 1—
In vivo alveolar endoscopy in smoking and nonsmoking healthy volunteers
Smokers Nonsmokers All subjects Subjects 17 24 41 Age yrs 37±16 46±13 42±15 (range 21–62) Male/female 9/8 10/14 19/22 FVC % predicted 101±16 104±15 103±16 Duration of alveolar procedure min 14±6 10±3 11±5 Alveolar mouth diameter μm 274±52 283±54 278±53 Measurements 149 125 274 Elastic fibre thickness μm 9.7±3.0 10.4±2.3 10±2.7 Measurements 445 464 909 Bronchus/Alexafluor fluorescence intensity ratio 0.20±0.11 0.12±0.07 0.14±0.09 Alveolus/bronchus fluorescence intensity ratio 0.86±0.69# 0.25±0.17# Microvessel diameter μm 95±47 89±56 90±50 Measurements 79 171 250 Subjects with alveolar fluorescent macrophages 16¶ 1¶ 17 Alveoli with fluorescent macrophages/total alveoli explored 77/85¶ 1/107¶ 78/192 Data are presented as n and mean±sd, with the exception of microvessel diameter, which is presented as median±interquartile range. FVC: forced vital capacity. #: p<0.0001, Mann–Whitney test; ¶: p<0.0001, Chi-squared test.
- Table 2—
Alveolar mouth and acinar microvessel sizes according to lung segments
Right lung Left lung RB3 RML RB6 RB7 RB8 RB9 RB10 LB 3-4-5 LB6 LB7+8 LB9 LB10 Alveolar areas reached n 9 43 15 15 18 14 8 28 6 22 8 6 Segments explored n 11 46 17 18 20 14 9 30 10 24 10 6 Alveolar mouth size μm 217±36# 289±59 289±40 244±52# 279±50 279±55 283±49 282±47 258±55 274±53 286±43 276±59 Measurements n 10 63 33 17 26 30 13 38 9 17 12 6 Acinar microvessel diameter μm 94 (59–123) 87 (60–123) 85.5 (69–102) 81 (71–132) 104 (68–143) 85 (69–120) 91 (54–145) 108 (93–132) 93 (66–210) 91 (81–121) 88 (68–103) 82 (58–106) Measurements n 13 57 12 13 37 24 12 16 8 39 12 7 Data are presented as mean±sd or median (interquartile range), unless otherwise stated. #: smaller alveolar ring entrances were found in RB3 and RB7 segments (p<0.001, ANOVA).
Supplementary data
Files in this Data Supplement:
- Supplementary methods and results
- Supplementary video - 3MB version
- Supplementary video - 7.5MB version
