Fig. 3: Observing 3D migration of neutrophils in the mouse brain in vivo.
a, Low-SNR images of neutrophil migration without denoising. b, Images denoised with DeepCAD-RT. c, Synchronized high-SNR images with tenfold fluorescence photons. Blue arrowheads point to the elongated tail of a migrating neutrophil. Magnified views of the yellow boxed region showing the morphological evolution of neutrophils in a 60-s time window. Red closed lines annotate the border of a neutrophil during migration. Neutrophils were labeled with a fluorescent-conjugated Ly-6G antibody. Each frame was integrated for 100 ms, and the entire time-lapse imaging session lasted 644 s; scale bars, 50 μm for the whole FOV and 10 μm for magnified views. d, x-t slices along the yellow dashed line in c of low-SNR raw data (left), DeepCAD-RT denoised data (middle) and corresponding high-SNR data with tenfold fluorescence photons (right); scale bars, 20 μm for x and 50 s for t. e, Box plots showing Pearson correlations of image slices along three dimensions (x-y-t) before and after denoising; x-y slice, N = 6,440; y-t slice, N = 512; x-t slice, N = 512. P values were calculated by one-sided paired t-test; ****P < 0.0001 for all comparisons. f, Denoising high-SNR data with DeepCAD-RT reveals subcellular dynamics of neutrophils. Retraction fibers are indicated with arrowheads; scale bar, 10 μm. g, Three-dimensional imaging of neutrophil migration in a 150 × 150 × 30 μm3 volume (15 planes) after acute brain injury. The raw noisy volume (left) and corresponding denoised volume (right) are visualized from the same perspective. Acute brain injury was induced by craniotomy. Neutrophils were labeled with a fluorescent-conjugated Ly-6G antibody (green channel). Blood vessels were stained with a WGA (magenta channel) dye. Because blood vessels are stationary, noise in the magenta channel was removed by averaging multiple frames; scale bar, 50 μm. h, Images of a single plane before (top) and after (bottom) denoising. DeepCAD reveals diffusion of the neutrophil population. Magnified views of yellow boxed regions are shown next to each image; scale bars, 50 μm for the entire FOV and 10 μm for magnified views.
