W plan apochromat 20 1.0 dic m27

To measure the spatial resolution of the mouse stimulator, we removed the lens of a Raspberry Pi camera chip (OV5647, Eckstein GmbH, Clausthal-Zellerfeld, Germany) and positioned it at the level of the recording chamber. Then, we projected UV and green checkerboards of varying checker sizes (2, 3, 4, 5, 10, 20, 30, 40, 60, 80, and 100 µm) through an objective lens (MPL5XBD (5x), Olympus, Germany) or through the condenser onto the chip of the camera (Figure 4a). For each checker size and LED, we extracted intensity profiles using ImageJ (Figure 4b) and estimated the respective contrast as $I_{Max}-I_{Min}$ (Figure 4c). To quantify the steepness of the transition between bright and dark checkers, we peak-normalised the intensity profiles and normalised relative to half-width of the maximum (Figure 4d). Next, we fitted a sigmoid to the rising phase of the intensity profile $y=K_0+K_1/(1+exp(-(x-K_2)/K_3\left)\right)$ and used $1/K_3$  as estimate of the rise time and as a proxy for ‘sharpness’ of the transitions between black and white pixels (Figure 4e).
To measure the difference in focal plane of UV and green LED due to chromatic aberration, we projected a 40 and 100 µm checkerboard through a 20x objective (W Plan-Apochromat 20×/1.0 DIC M27, Zeiss, Oberkochen, Germany) onto the Raspberry Pi camera (see above).

For all imaging experiments, we used MOM-type two-photon (2P) microscopes (designed by W. Denk, MPI, Heidelberg; purchased from Sutter Instruments/Science Products, Hofheim, Germany). For image acquisition, we used custom software (ScanM by M. Müller, MPI Neurobiology, Munich, and T.E.) running under IGOR Pro 6.3 for Windows (Wavemetrics, Lake Oswego, OR). The microscopes were equipped each with a mode-locked Ti:Sapphire laser (MaiTai-HP DeepSee, Newport Spectra-Physics, Darmstadt, Germany; or Chameleon Vision-S, Coherent; Ely, UK), two fluorescence detection channels for eCFP (FRET donor; HQ 483/32, AHF, Tübingen, Germany) and citrine (FRET acceptor; HQ 538/50, AHF) or GCaMP6f (ET 525/70 or ET 525/50, AHF), and a water immersion objective (W Plan-Apochromat 20×/1.0 DIC M27, Zeiss, Oberkochen, Germany). The excitation laser was tuned to 860 nm and 927 nm for TN-XL (eCFP) in mouse and GCaMP6f in zebrafish, respectively. Time-lapsed image series were recorded with 64 × 16 pixels (at 31.25 Hz) or 128 × 64 (at 15.625 Hz). Detailed descriptions of the setups for mouse (Euler et al., 2019 (link); Euler et al., 2009 (link); Franke et al., 2017 (link)) and zebrafish (Zimmermann et al., 2018 (link)) have been published elsewhere.

We used a MOM-type two-photon microscope (designed by W. Denk, MPI, Heidelberg; purchased from Sutter Instruments/Science Products)^{73 (link)}. In brief, the system was equipped with a mode-locked Ti:Sapphire laser (MaiTai-HP DeepSee, Newport Spectra-Physics), two fluorescence detection channels for iGluSnFR/OGB-1 (HQ 510/84, AHF/Chroma) and SR101/tdTomato (HQ 630/60, AHF), and a water immersion objective (W Plan-Apochromat ×20 /1.0 DIC M27, Zeiss). The laser was tuned to 927 nm for imaging iGluSnFR, OGB-1 or SR101, and to 1000 nm for imaging tdTomato. For image acquisition, we used custom-made software (ScanM by M. Müller and T.E.) running under IGOR Pro 6.3 for Windows (Wavemetrics), taking time-lapsed 64 × 64 pixel image scans (at 7.8125 Hz) for OGB-1 imaging in the GCL and 128 × 128 pixel image scans (at 3.9 Hz) for glutamate imaging in the outer plexiform layer (OPL). For vertical glutamate imaging in the IPL, we recorded time-lapsed 64 × 56 pixel image scans (at 11.16 Hz) using an electrically tunable lens (for details, see ref. ^{35 (link)}). For high resolution images, 512 × 512 pixel images were acquired. The positions of the fields relative to the optic nerve were routinely recorded.