Laser injury assessed with commercial SLO and OCT. (A) 488 nm light is focused onto the photoreceptor outer segments using AOSLO. Created with Biorender.com. (B) 30° SLO images of NIR reflectance, blue reflectance and fluorescein angiography of a mouse retina 1 day after laser exposure. Three focal planes are shown. NIR and blue reflectance reveal small hyperreflective regions below the superficial plane. Fluorescein reveals intact vasculature with no sign of leakage. Arrows indicate regions with imparted laser damage (1-4). (C) OCT B-scans passing through laser-exposed regions indicated in B. Exposures produced a focal hyperreflective band within the ONL with adjacent retina appearing healthy. OCT images were spatially averaged (∼30 µm, 3 B-scans). Scale bars = 200 µm horizontal, 200 µm vertical.

Laser damage temporally tracked with AOSLO and OCT. Laser-exposed retina was tracked with OCT (A), confocal (B) and phase-contrast (C) AOSLO for baseline, 1, 3, 7 day and 2 month time points. OCT and confocal AOSLO display a hyperreflective phenotype that was largest/brightest at 1 day and became nearly invisible by 2 months. Dashed oval indicates region targeted for laser injury. Phase-contrast AOSLO revealed disrupted photoreceptor soma’s 1 day after laser injury. OCT images were spatially averaged (∼30 µm, 8 B-scans). Scale bars = 40 µm, OCT image vertical scale bar = 100 µm.

Retinal histology confirms photoreceptor ablation and preservation of inner retinal cells. Cross sectional view (A) and en-face (B+C) images of DAPI-stained whole-mount retinas (5 mice) at laser injury locations over time. By 1 day, ONL becomes thicker at lesion location, but thinner by 3 and 7 days. By 2 months, the ONL appeared similar to that of control. The inner (B, solid rectangle) and outer (C, dashed rectangle) stratum of ONL show axial differences in ONL loss. Most cell loss was seen in the outer aspect of the ONL (C). Scale bars = 40 µm. (D) Cross section of DAPI-stained retina displaying INL and ONL regions for quantification. Each analysis region was 50 µm across and encompassed the entire INL or ONL. (E) En-face images show 50 µm diameter circles used for analysis. (F) Nuclei density for control and post-injury time points. ONL nuclei were reduced at 3 and 7 days (p = 0.17 and 0.07 respectively) while INL density remained stable (n = 10 mice, 3 unique regions per time point). Error bars display mean ± 1 SD.

Microvascular perfusion unchanged after laser damage. A single location tracked over time and at three vascular plexuses using AOSLO. Motion-contrast images were generated from confocal videos to reveal vascular perfusion status. Retinal vasculature remained perfused for all time points tracked and at all depths. White oval indicates damage location. Scale bar = 40 µm.

Microglial response 1 day after laser injury imaged in vivo with fluorescence SLO and AOSLO. (A) Left: Deep-focus NIR SLO fundus image (55° FOV) of laser-injured retina. White arrowheads point to damaged locations showing hyperreflective regions. Inset scale bar = 40 µm. Right: Fluorescence fundus image from same location. Fluorescent CX3CR1-GFP microglia are distributed across the retina and show congregations at laser-damaged locations. Scale bar = 200 µm. (B) Magnified SLO images of microglia at laser-damaged and control locations (indicated in A, right, white boxes). Control location displays distributed microglial whereas microglia at the lesion location are bright and focally aggregated. (C) Fluorescence AOSLO images show greater detail of cell morphology at the same scale. In control locations microglia showed ramified morphology and distributed concentration whereas damage locations revealed dense aggregation of many microglia that display less ramification. Scale bars = 40 µm.

Microglial response to laser injury tracked with AOSLO. Simultaneously acquired NIR confocal and fluorescence AOSLO images across different retinal depths. Data are from one CX3CR1-GFP mouse tracked for 2 months. Microglia swarm to hyperreflective locations within 1 day. Microglia maintain an aggregated density for days and resolve by 2 months after damage. Scale bar = 40 µm.

Neutrophil morphology imaged in vivo using AOSLO. (A) Phase-contrast, motion-contrast and fluorescence AOSLO reveal the impact of passing neutrophils on single capillaries. A rare and exemplary event shows a neutrophil transiently impeding capillary blood flow for minutes in healthy retina. Scale bar = 40 µm. (B) In-vivo AOSLO and ex-vivo fluorescence microscopy show neutrophils in two states. Neutrophils within capillaries displayed elongated, tubular morphology. Extravasated neutrophils were more spherical. Bottom images show extravasated neutrophils in response to LPS model for comparison (not laser damage model). Scale bar = 20 µm.

Retinal damage location tracked with AOSLO does not show accumulation of neutrophils. A single retinal location was tracked in a Catchup mouse from baseline to 2 months after lesion. Location of the lesion is apparent at 1 and 3 days post injury with diminishing visibility after 1 week. Despite the ability to detect neutrophils (Figure 7), we did not observe stalled, aggregated or an accumulation of neutrophils at any time point evaluated. This evaluation was confirmed at multiple depths ranging from the NFL to the ONL of the retina in vivo. Scale bar = 40 µm.

Neutrophil behavior under laser injury, as observed through ex vivo confocal microscopy, remains unresponsive despite marked microglial activation (A) En-face max intensity projection images of inner and outer (separated by approximate INL center) retinal microglia/neutrophils in Ly-6G-647-stained CX3CR1-GFP retinas. Microglia display focal aggregation in the outer retina for 1, 3 and 7 day time points that is resolved by 2 months. Neutrophils do not aggregate or colocalize to the injury location at any time point. Z-stacks were collected from 5 mice for the indicated time points. (B) Cross-sectional views of en-face z-stacks presented in A, including DAPI nuclear label. White dotted line indicates 100 µm region expanded below. Microglia migrate into the ONL by 1, 3 and 7 days post-laser injury and return to an axial distribution similar to that of control by 2 months. The few neutrophils detected remained within the inner retina. Scale bars = 40 µm. (C) Orthogonal view of DAPI-stained retina with Ly-6G-647-labelled overlay 1 day post-laser-injury. In a rare example, 2 neutrophils are found within the IPL/OPL layers despite a nearby outer retinal laser lesion. Scale bar = 20 µm. (D) Magnified 3D cubes representing cell 1 and 2 in C. Cell 1 displays pill-shaped morphology and cell 2 is localized to a putative capillary branch-point. Each are confined within vessels suggesting they do not extravasate in response to laser injury.

Quantification of neutrophils in laser damaged retinas assessed with ex vivo confocal microscopy over a wide-field. (A) Representative image displays neutrophils quantified using large-field (796 x 796 µm) z-stacks for control or 1 day after injury time points. In both control and laser injured retinas, neutrophils were sparse and confined to locations within capillaries suggesting they were the native fraction of circulating neutrophils at time of death. Inset displays expanded image of a single neutrophil. Scale bar = 200 µm. (B) Neutrophils quantified and displayed as the number of neutrophils per retinal area. The difference in number of neutrophils in control vs lesioned retinas was not statistically significant (p = 0.19). Error bars display mean + 1 SD.

Lesion location tracked from minutes to 1 day with OCT. After baseline OCT acquisition, OCT was performed every 5-7 minutes for one hour after 488 nm light exposure. 6 hour and 1 day time points were subsequently acquired. A band of hyperreflectivity forms near the OPL/ONL interface within 30 minutes of 488 nm light exposure. Hyperreflective band, spreads deeper into the ONL within ∼1 hour. OCT images were spatially averaged (∼30 µm, 8 B-scans). Scale bar = 40 µm horizontal, 100 µm vertical.

Measurement of single-cell blood flux after laser damage using phase contrast AOSLO. Mouse 1: (A) The vascular plexus corresponding to IPL (cyan) and OPL (red) were targeted for flux determination. Blood cell flux was measured for 2 capillaries within the same field, at different depths. Arrows show the location for repeated line scan acquisitions. (B) RBC flux images were tracked up to 7 days post-damage. Scale bars = 10 ms, horizontal and 5 µm vertical. (C) Capillary flux measurements over 7 days. Despite the outer capillary displaying higher flux, both inner and outer capillaries changed synchronously for each time point. (D) Correlation of inner and outer capillary flux. Linear regression model displays a weak positive correlation (black dotted line). Mouse 2: (E) Left: Representative 55° SLO image showing regions targeted for capillary flux measurement. One region was subject to 488 nm laser damage and the other was left unlasered (Control). Scale bar = 200 µm. Right: Capillaries targeted for blood cell flux measurement. Arrows show the location for repeated line scan acquisitions. Scale bar = 40 µm. (F) RBC flux images were tracked up to 2 months post-damage. Scale bars = 10 ms horizontal and 5 µm vertical. (G) Capillary flux remained similar at lesion and control locations over all time points assessed. Gray shaded regions indicate range for normal capillary flux in the healthy C57BL/6J mouse (Dholakia et al 2022). (H) Correlation of flux in lesion and control locations. Linear regression model displays a positive correlation (black dotted line).

Hyperreflective appearance emerges before microglia swarm to damage location. AOSLO confocal and fluorescence images were acquired for baseline, 30, 90 minute and 1 day post-laser exposure. The hyperreflective phenotype appeared within 30 minutes post damage. Microglia were not found to aggregate until ∼1 day after. Scalebar = 40 µm.

Neutrophil/microglial response to laser injury tracked with ex vivo confocal microscopy. Simultaneously acquired GFP-positive microglia and Ly-6G-647-positive neutrophils were imaged with confocal microscopy in 5 CX3CR1-GFP mice. En-face images for several retinal depths are displayed. By 1, 3 and 7 days post-lesion, microglia have migrated into the outer retina, many appearing amoeboid and displaying fewer laterally-branching projections. Despite the deep microglial response, neutrophils stay within the inner retina and are not found in the avascular outer retinal layers. Scale bar = 40 µm.

Microglial PR phagosomes in the outer retina assessed with ex vivo confocal imaging. (A) En-face images of outer ONL in a DAPI-stained CX3CR1-GFP mouse 3 days post-laser-injury (top row). Microglia have infiltrated deep into the ONL and several PR phagosomes were identified. White arrows indicate locations for a single microglia (i), PR (ii) and PR phagosome (iii). These locations were expanded and displayed below. Microglia exhibited a heterogeneous nuclear staining pattern while PR nuclei exhibited homogenous DAPI fluorescence pattern. PR’s displayed this pattern regardless of whether they were within a microglial phagosome or not. Top scale bar = 20 µm, bottom scale bar = 2 µm. (B) A finely-sliced (0.1 µm step size) outer retinal z-stack of DAPI-stained CX3CR1-GFP retina was used to quantify the average nuclear volume for infiltrated microglia (n = 14 nuclei) and PR’s (n = 20 nuclei) for the same lesion site presented in A. On average, microglia had a statistically significant (p < 0.001) nuclear volume that was >3x that of PR’s. These measurements allowed us to discriminate microglial somas from PR phagosomes. Error bars display mean + 1 SD. (C) Cross sections of DAPI-stained outer retina in CX3CR1-GFP mice for 1, 3 and 7 days post-laser injury (n=3 mice). 3 representative planes (X-Z) through the lesion are displayed for each time point. Microglia form PR phagosomes within the ONL and microglial processes were seen extended into the PR inner/outer segment layer. Arrows label various morphological features seen at lesion sites: microglial somas (yellow), diving microglial process (violet), PR phagosome (red), microglial inner/outer segment process (cyan). Scale bar = 20 µm.