Fig. 6.

Cleared tissue digital scanned light-sheet microscopy (C-DSLM) imaging of rat lungs. a Rat lungs (day 14) were collected, inflated, fixed, cleared using passive CLARITY (PACT) (scale bar—5 mm). b Individual image plane of epithelium (blue) and endothelium (red) within rat lung from a 1.3 mm × 1.3 mm × 6 mm axial image (scale bar—250 μm) acquired using the ×10, NA 0.28 detection objective. (scale bar—250 μm) The light-sheet full-width at half-maximum (FWHM) was adjusted to ∼10 μm to yield an approximate Rayleigh length of 700 μm before Gaussian beam scanning. Each axial step was ∼1 μm. c Expanded image of highlighted area (white box) in b showing distal lung structure. Alveolar structures were clearly marked by both airway (blue), blood vessels (red), and overlap between the two networks (magenta) (scale bar—50 μm). d 2D histology of distal lung imaged using white light and a ×20 air immersion objective (scale bar—200 μm). e 3D reconstruction of c. The true complexity of the distal lung was lost in traditional histology but can be clearly seen using tissue clearing and light-sheet imaging. Supplementary Movie 5 shows a volumetric rendering of this region. f Verification of C-DSLM distal lung results using Mean Linear Intercept (MLI) to determine the chord length (L _m). We created a new 3D MLI algorithm that imposes a three-dimensional grid (vs the traditional lines for planar MLI) and calculates nearest-neighbor intersections across the volume measurement. Serial sectioning and histological staining measured by wide-field microscopy (n = 23 individual histology slices) versus cleared and intact lung lobe measured by C-DSLM (n = 5 entire lungs, 5000 individual image planes). Error bars are standard deviation (SD). Mean L _m values for histology and C-DSLM L _m did not significantly differ. The larger C-DSLM SD was due to both our inclusion of 3D information and proximal lung blood vessels, leading to a larger spread in calculated L _m