3D microscopy and deep learning reveal the heterogeneity of crown-like structure microenvironments in intact adipose tissue

Figures

• 

  Fig. 1. Adipose tissue clearing, labeling, and fluorescence microscopy for visualization of macrophage and CLS features. (A) Bright-field images of adipose tissue punches before and after treatment with four clearing techniques. All images share the same scale bar of 5 mm. (B) Bright-field images of intact perirenal adipose tissue before and after iDISCO clearing. Both images share the same scale bar of 1 cm. (C and D) Bright-field images of freshly dissected perirenal adipose tissue before clearing from both obese and lean mice and corresponding low-magnification light sheet microscopy images after iDISCO clearing. Tissues are stained with YO-PRO-1 nuclear dye (red) and a fluorescently labeled antibody against the macrophage F4/80 antigen (green). Scale bars, 10 mm for bright-field images and 5 mm for fluorescence images. (E and F) Zoomed light sheet microscopy images and (G and H) confocal fluorescence images for obese and lean tissues. Scale bars, 200 μm (E and F) and 20 μm (G and H). All fluorescence images are 3D projection images.

• 

  Fig. 2. CLS segmentation with Mask R-CNN algorithm. (A) Workflow of Mask R-CNN for CLS detection and segmentation. ROI, region of interest. (B) Free-response receiver operating characteristic (FROC) curve for testing results with Mask R-CNN with an average number of 100 ground truth CLSs. (C and D) Typical 3D macrophage-labeled light sheet images and respective segmentation images for adipose tissue from (C) obese and (D) lean mice. All images share the same scale bar of 200 μm. (E and F) Distributions and four-component Gaussian fits for CLSs in perirenal adipose tissues in (E) obese and (F) lean mice. Components are given in the legends together with fractional contribution to the total counts in parentheses. (G) CLS and (H) cell number densities across adipose tissue in both obese and lean mice (n = 4). Asterisks indicate the following: *P = 0.0143 and **P = 0.0034, Student’s t test.

• 

  Fig. 3. Macrophage identification in intact adipose tissues and in CLSs. (A) Representative images of obese and lean adipose tissues showing raw nuclear image, nuclear segmentation, nuclei of cells classified as macrophages overlaid on CLS segments, and macrophages within CLSs. (B and C) Histograms show F4/80 intensity distribution across cells in obese and lean adipose tissues, in addition to six component Gaussian fittings. Plots show 10 million cells in obese adipose tissue and 5 million cells in lean adipose tissue. a.u., arbitrary units. (D and E) Cell populations in obese and lean adipose tissues, including macrophages (MΦ) and other cells (non-MΦ) within or not within CLSs. (F) Cell counts within CLSs from adipose tissues of lean and obese mice (*Mann-Whitney U < 0.0001). (G) Volumetric density of cells within each CLS calculated by dividing cell number by CLS volume (**Mann-Whitney U = 0.0038). In (F) and (G), boxes indicate 25th/75th percentile, black lines are mean values, whiskers are SDs. (H and I) Density plots of CLS volume versus cell number in adipose tissue of obese and lean mice. Linear and quadratic fits are shown with indicated coefficients of determination (R2).…

• 

  Fig. 4. Structural heterogeneity of CLSs. (A to F) Representative 3D light sheet microscopy images of CLSs in adipose tissue showing both 3D projections and slices. Scale bars, 50 μm. The CLS volume (V), diameter (D), cell number (N), sphericity (S), oblate ellipticity (O), and prolate ellipticity (P) are listed. (G) Unsupervised hierarchical clustering of the CLS parameters in adipose tissue of obese and lean mice. The corresponding cluster of the representative CLSs in (A to F) is (A and B), C4; (C and D), C5; and (E and F), C7. (H) Density plots of CLS ellipticity for oblate (O) and prolate (P) shape classification versus volume in adipose tissue of obese and lean mice. (I and J) Mean oblate and prolate ellipticity is shown with 95% confidence intervals (shaded lines) for indicated volumes of lean CLSs (I) and obese CLSs (J) with rolling average of 1.5 × 105 μm3. (K) Mann-Whitney U test for comparison of oblate and prolate ellipticity between obese and lean CLSs with rolling 1.5 × 105 μm3 increment data binning across the indicated volumes.