Figure 2. SLOT imaging and screening for ROI.

(a) Volumetric imaging of the whole lobe with SLOT. The CRISTAL sample is immersed in a cuvette filled with immersion oil and mounted to a stage allowing for stepwise rotation. An f-theta objective forms a focused laser beam that is scanned through the sample to generate 2D projection images. Synchronized with the scanning process, transmitted light is recorded with a photo diode (PD), while fluorescence is simultaneously measured from below via photomultiplier tube (PMT). (b) Projection images of the lobe from one viewing angle, each covering a region of 13 × 15.5 mm. (c) By measuring a full revolution of the sample at step angles of 0.24°, two projection data sets with fluorescence and absorption data from 1,500 viewing angles are acquired. In order to generate 3D data stacks containing the whole sample, filtered back projection algorithms are applied to both sets. (d) Single tomograms after reconstruction of fluorescence and absorption channel. (e) Rendered volume overlay of absorption (red) and fluorescence (green) channels. (f) Reslice through the stack at the cutting plane indicated in e demonstrates alveolar resolution at different depths. Typical phenotype of fibrosis like parenchyma tissue thickening is visible. (g,h) Identification of ROI with increased tissue remodeling and collagen deposits is achieved by segmentation. Here, fibrotic regions (green) and normal parenchyma (blue) in the bleomycin-treated lung can easily be identified and compared with the untreated control lung. Scale bars, 1 mm (b,f,g,h), 500 μm (d).