A1rmp microscope

HEK293-Kir2.1 cells were transfected as described above. Cells were imaged with an A1R MP+ microscope (Nikon, Japan) fitted with 20× 0.75NA dry objective, a 525/50 nm filter, gallium arsenide phosphide (GaAsP) detectors, and a titanium:sapphire Chameleon Ultra I laser (Coherent, Santa Clara, CA) with a 80 MHz repetition rate and a pulse width of 140 fs (measured at 800 nm). The laser was tuned between 700 nm and 1040 nm, keeping the laser power at 10 mW across the spectrum. However, this system did not pre-compensate laser pulses for dispersion in the microscope optical path. Pulse width is therefore expected to vary with wavelength (Müller et al., 1998 (link)), thereby impacting two-photon excitation absorption efficiency and distorting excitation spectra over those acquired by a pre-compensated system. Laser scanning was performed using galvanometric mirrors. Each image pixel was sampled with a dwell time of 12.1 µs. To evaluate the impact of photobleaching, we acquired images at 900 nm both before and after each wavelength scan. We also compared spectra obtained by scanning from 700 nm to 1040 nm to those produced in the reverse direction (1040 nm to 700 nm). Both methods demonstrated that photobleaching was negligible, and we therefore did not correct for photobleaching.

Channelrhodopsin activation was achieved with a custom LED device including an optic fiber cable mounted on a 485 nm blue LED (Cree Lighting Inc., Durham, NC) connected to a SLB-1200-1 universal LED driver (Mightex, Pleasanton, CA). The end of the optic fiber cable was placed a few millimeters away from the cells with a MP-285 micromanipulator (Sutter Instrument, Novato, CA). Light intensity was calibrated at 0.38 mW/mm² using a PM20A optical power meter (Thorlabs, Newton, NJ). Light pulses were Transistor-Transistor Logic (TTL) triggered with pClamp10.6 software using the analog-input of the LED driver. For some sets of experiments, channelrhodopsin was stimulated using a Spectra X (Lumencor, Beaverton, USA) directly coupled to the epifluorescent port of the Nikon A1R-MP microscope. Light pulses were TTL triggered with patchmaster software.

The dynamics of DiD-positive leukocytes were observed using an intravital 2-photon microscope. A female 3-week-old WKY rat was injected with 50 μg TF78 and DiD-labeled ASCs (1 × 10⁶ cells) on day 0. On day 2, the rat was anesthetized with a mixture of medetomidine midazolam and butorphanol and injected with 0.25 μg/g body weight FITC anti-rat CD45 (Biolegend, 202226) and 0.01 mg/g body weight rhodamine-70k dextran. Then, the spleen was exteriorized with a lateral incision and extended over the imaging platform. Time-lapse images were acquired at 30 s per frame using a Nikon A1RMP microscope.

Two-photon functional imaging in the retina was performed using a Nikon A1R MP microscope equipped with a 4-channel GaAsP NDD and an Apochromat 25X/1.1 NA water-immersion objective (Nikon). Excitation was provided by a Chameleon Ultra II Mode-locked titanium-sapphire laser (Coherent) tuned to 930 nm. Time-series of visually evoked calcium responses were acquired at a rate of 4 Hz and 0.248 × 0.248 μm resolution (512 × 256 pixels). Following activation of the laser scanning, we waited 60 seconds before starting the visual stimulation to ensure the retina adapted to the background light level caused by the multi-photon laser. 4 dpf crystal Tg(elavl3:GCaMP6f) larvae were first paralysed for 10–15 minutes in α-bungarotoxin (1 mg/ml; Biotium) prepared in Danieau solution. Subsequently, larvae were immobilized in 2% low melting point agarose (Sigma) and mounted on a raised custom-made glass platform with the dorsal side up (45° angle tilt) and one eye facing an LCD screen (see Visual stimulation) that was placed underneath a custom-made Danieau-filled chamber. Imaging was performed in the afternoon (1–8 pm).