Journal: Cell Reports Methods

Article Title: Projection targeting with phototagging to study the structure and function of retinal ganglion cells

doi: 10.1016/j.crmeth.2026.101308

Figure Lengend Snippet: Identification of transduced RGCs using in vivo fluorescence fundoscopy and confocal imaging (A–O) Fluorescence fundus images are shown from two example rats. The schematic for rat one illustrates the left eye at post-operative days (PODs) (A) 5, (C) 9, (E) 13, (G) 17, (I) 21, and (K) 27. Red lines depict the intraocular vasculature used to align fundus images across days. Insets (B, D, F, H, J, and L) show magnified views of regions with prominent labeling, as indicated by the dashed white rectangles (A, C, E, G, I, and K, respectively). Examples of transduced RGCs are indicated with Roman numerals (i–xii), several of which are present across multiple imaging time points. The schematic for rat two (M–O) illustrates an example of expression longevity starting from POD 14 through POD 146. Note: variations in overall fluorescence intensity across time points are likely caused by differences in fundus camera positioning between imaging sessions; we therefore do not attribute these changes to fluctuations in viral expression. The spatial pattern of labeled RGCs remains stable, indicating persistent expression. (P) Injection of AAV2retro-CAG-ReaChR-mCitrine into the left SC resulted in labeling across tectal layers, as indicated by the arrows. (Q) This injection resulted in fluorescence fundoscopic detection of mCitrine-positive RGC labeling within the nasal quadrant of the right eye. (R) A low-magnification confocal photomicrograph of the eyecup following anti-GFP immunofluorescent amplification. The dashed box delineates the region shown in (S). (S) A confocal photomicrograph shown at the estimated scale as the fluorescence fundus image in (Q). Alignment of these images was achieved using the major blood vessels. Roman numerals i and ii identify examples of vessel bifurcation used to match the images from fluorescence fundoscopy with those from confocal imaging. (T–W) Examples of retrogradely transduced RGCs are depicted with arrows in aligned fluorescence fundoscopy images (T and V) and corresponding confocal images (U and W). Solid white arrows mark RGCs clearly visible in both imaging modalities. A hollow arrow in (V)/(W) marks an RGC that is evident in the confocal image (W) but not clearly distinguishable in the fundus image (V), highlighting the potential bias of fluorescence fundus imaging toward more brightly labeled cells. .

Article Snippet: Goat Anti-GFP , Rockland , Cat# 600-101-215; RRID: AB_218182.

Techniques: In Vivo, Fluorescence, Imaging, Labeling, Expressing, Injection, Amplification