Fig. 1

Characterization of the Cx3cr1–Dendra2 mouse. A Immunohistochemistry of Cx3cr1^D2/D2 retina showing Iba1⁺ microglia staining (top) is concurrent with Cx3cr1–Dendra2 expression (amplified using anti-GFP antibody, middle) and displays normal microglia morphology. Scale 500 µm; from a female mouse, n = 3 retina (3 mice). B Merged image of live retinal explants from a male Cx3cr1^D2/D2 mouse showing microglia with intrinsic unconverted gD2 fluorescence under 488 nm light (left) and a single photoconverted cell expressing rD2 under 560 nm light (right). Scale 100 µm, n = 6 retinas (5 mice). C Schematic of photoconversion. Naïve microglia shown in green; triangles indicate gD2. Following noninvasive 405 nm light exposure (center), gD2 permanently converts to rD2 (red squares), shifting microglia fluorescence to a red/orange color. Over time, protein turnover causes an extinction of rD2 and gain of gD2 (right). Insets show gD2 and rD2 structure. D D2 spectral analysis of the photoconverted cell in B shows that prior to photoconversion, the combined emission spectrum from 488 and 560 nm excitation has a strong gD2 primary peak at 505 nm and a slight rD2 secondary peak at 571 nm (green trace, time 0 s). Exposure to 2 min of 405 nm light decreases the gD2 peak and sharply increases the rD2 peak (red trace), reflecting rapid photoconversion (time course shown in multicolored traces). E D2 conversion rates of the photoconverted cell in B indicate that the 488 nm laser caused gD2 photobleaching prior to 405 nm onset but did not cause appreciable photoconversion (green trace). Onset of the 405 nm laser at t = 0 caused a rapid rise in rD2 (red trace) and a decrease in gD2