We used a MOM-type two-photon microscope (designed by W. Denk, MPI, Martinsried; purchased from Sutter Instruments/Science Products, Hofheim, Germany). Design and procedures were described previously20 (link). In brief, the system was equipped with a mode-locked Ti:Sapphire laser (MaiTai-HP DeepSee, Newport Spectra-Physics, Darmstadt, Germany) tuned to 927 nm, two fluorescence detection channels for OGB-1 (HQ 510/84, AHF/Chroma Tübingen, Germany) and SR101 (HQ 630/60, AHF), and a water immersion objective (W Plan-Apochromat 20x/1,0 DIC M27, Zeiss, Oberkochen, Germany). For image acquisition, we used custom-made software (ScanM, by M. Müller, MPI, Martinsried, and T.E.) running under IGOR Pro 6.3 for Windows (Wavemetrics, Lake Oswego, OR), taking 64 × 64 pixel image sequences (7.8 frames/s) for activity scans or 512 × 512 pixel images for high-resolution morphology scans.
For light stimulation, we focused a DLP projector (K11, Acer) through the objective, fitted with band-pass-filtered light-emitting diodes (LEDs) (“green”, 578 BP 10; and “blue”, HC 405 BP 10, AHF/Croma) that roughly match the spectral sensitivity of moose M- and S-opsins. LEDs were synchronised with the microscope’s scan retrace. Stimulator intensity (as photoisomerisation rate, 103 P*/s/cone) was calibrated as described previously52 (link) to range from 0 (LEDs off) to 10.8 and 9.9 for M- and S-opsins, respectively. Due to two-photon excitation of photopigments, an additional, steady illumination component of ~104 P*/s/cone was present during the recordings (for detailed discussion, see22 (link)). For all experiments, the tissue was kept at a constant intensity level (see stimuli below) for at least 30 s after the laser scanning started before light stimuli were presented. Four types of light stimulus were used (Fig. 1b , top): (i) Full-field “chirp” stimulus consisting of a bright step and two sinusoidal intensity modulations, one with increasing frequency and one with increasing contrast, (ii) 0.3 × 1 mm bright bar moving at 1 mm/s in 8 directions19 (link), (iii) alternating blue and green 3 s flashes, and (iv) binary dense noise, a 20×15 matrix with 40 μm pixel-side length; each pixel displayed an independent, perfectly balanced random sequence at 5 Hz yielding a total running time of 5 minutes for receptive field (RF) mapping. In some experiments, we used in addition dark moving bars (like (ii) but contrast-inversed), and stationary bright or dark 0.2 × 0.8 mm bars flashed for 1 s in 6 orientations (see Extended Data Fig. E7g–i ). Except for (iii), stimuli were achromatic, with matched photo-isomerisation rates for M- and S-opsins.
For light stimulation, we focused a DLP projector (K11, Acer) through the objective, fitted with band-pass-filtered light-emitting diodes (LEDs) (“green”, 578 BP 10; and “blue”, HC 405 BP 10, AHF/Croma) that roughly match the spectral sensitivity of moose M- and S-opsins. LEDs were synchronised with the microscope’s scan retrace. Stimulator intensity (as photoisomerisation rate, 103 P*/s/cone) was calibrated as described previously52 (link) to range from 0 (LEDs off) to 10.8 and 9.9 for M- and S-opsins, respectively. Due to two-photon excitation of photopigments, an additional, steady illumination component of ~104 P*/s/cone was present during the recordings (for detailed discussion, see22 (link)). For all experiments, the tissue was kept at a constant intensity level (see stimuli below) for at least 30 s after the laser scanning started before light stimuli were presented. Four types of light stimulus were used (
