Fig. 2: Microglial navigation through complex brain tissue is revealed by confocal shadow imaging.

A Z-stack of confocal shadow images (AlexaFluor 594, 200 μM) of CA1 area of hippocampus in an organotypic brain slice from a transgenic mouse line (CX3CR1-EGFP), where all microglial cells are fluorescently labeled with EGFP (see also SI Movie 5; representative of 4 independent experiments). Top left panel shows maximum-intensity projection of only EGFP-labeled microglia. Other panels show overlay of EGFP and shadow image for three different optical sections (16.5, 20.5, and 23 µm below the slice surface). Scale bars, 20 μm. B Zoom inset of overlay of EGFP and shadow image (20.5 µm depth), with web-Knossos-based manual segmentation of putative dendrite, and 3D projection (10° horizontal rotation) of segmented dendrite enwrapped by EGFP-positive microglial process (see also SI Movie 6). Scale bars, 20 μm. C Time series of confocal shadow images (AlexaFluor 594, 200 μM) showing labeled microglia cells (CX3CR1-EGFP) reacting to a laser lesion (blue bolt), rapidly extending their fine processes towards the site of the lesion (see also SI Movie 7; representative of 8 independent experiments). Scale bar, 20 μm. D Representative manual traces of EGFP-positive microglial processes extending towards the site of laser lesion, impeded by either cell bodies (magenta) or by neuropil (blue). Scale bar, 10 μm. Plot of mean distance traveled (µm; mean ± SEM) by these processes over time (seconds; n = 29 microglial processes through neuropil, n = 14 microglial processes around cell bodies, from 8 independent experiments, thin lines = traces of individual processes). Two-way repeated measures ANOVA with Bonferroni post hoc correction (time: F(2.419, 99.19) = 600.5, ****p < 0.0001, process path: F(1, 41): 58.01, ****p < 0.0001, interaction: F(15, 615) = 37.23, ****p < 0.0001). Source data are provided as a Source data file. E Representative image (left) from 20 min after lesion induction showing EGFP-positive microglial processes with phagocytic cups labeled (green). Scale bar, 10 μm. Representative COSHI image (right, grayscale) from the identical timepoint with overlay of intact (dye-negative; blue) and lysed (dye-positive; red) tissue, as well as phagocytic cups (green) and developing lesion area (white dashed line). Contents of phagocytic cups are identified by overlay (cyan—intact; yellow—lysed). Pie chart indicates the relative contents of phagocytic cups over 8 lesion time series (n = 130 phagocytic cups examined during 8 independent experiments). Source data are provided as a Source data file.