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Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo

Fig. 5

Microglial response to localized damage. A Composite widefield images showing naïve microglia (green) and photoconverted microglia (red) in the retina prior to focal NIR damage (pre-damage) and the subsequent 1 day to 3 weeks in the same female mouse (n = 1 retina). Cells were photoconverted in an area of ~ 365 × 365 µm (9 J/cm2, dashed white line); retinal damage was induced at the injury site (yellow, ~ 150 µm diameter). All detectable rD2 microglia migrated to the injury field within 24 h, progressively forming a dense, bright cluster of cells over the first 3 days. Scale 300 µm. B Quantified intensity of gD2 (green, top trace) and rD2 (red, bottom trace) fluorescence in the injury site shown in A over time. The gD2 signal was initially bright against the background but lost nearly twofold intensity after photoconversion (lower point at t = 0); conversely, the rD2 signal increased nearly threefold after photoconversion (upper point at t = 0), peaking 24 h after damage, then falling to baseline levels by 1 week. C, D Magnified views of the gD2 (488 nm excitation) and rD2 (561 nm excitation) fluorescent images at each timepoint within the injury locus. Scale 100 µm. E OCT imaging was performed simultaneously to monitor the integrity of retinal layers and the changes in light scattering characteristic of retinal damage in the same mouse as A. Both OCT B-scans (top, optical sections) and en face images (bottom) through the injury locus reveal the increase in light scattering in the outer nuclear layer (ONL) evoked by the NIR laser damage (Post-damage). Note that prior to NIR laser exposure (Pre-damage) retinal layers appeared normal, confirming that the prior 405 nm light exposure used for D2 photoconversion did not cause photoreceptor damage. F OCT B-scans (top) and en face images (bottom) for subsequent timepoints matched to corresponding SLO in A. The localized damage from the NIR exposure was most extensive at 1 day and nearly resolved by 3 weeks. B-scan scale 100 µm horizontal, 50 µm vertical; en face scale 300 µm. GC ganglion cell layer, INL inner nuclear layer, OS outer segments, RPE retinal pigment epithelium

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