Pmt2101

It is taken with a commercial multiphoton microscope with both 2P and 3P light path (Bergamo II, Thorlabs). A high numerical aperture (NA) water immersion microscope objective (Olympus XLPLN25XWMP2, 25 X, NA 1.05) is used. For GFP and THG imaging, fluorescence and THG signals are separated and directed to the detector by a 488 nm dichronic mirror (Di02-R488, Semrock) and 562 nm dichronic mirror (FF562-Di03). Then the GFP and THG signals are further filtered by a 525/50 nm band-pass filter (FF03-525/50, Semrock) and 447/60 nm (FF02-447/60, Semrock) band-pass filter, respectively. The signals are finally detected by GaAsP photomultiplier tubes (PMTs) (PMT2101, Thorlabs).

Ca²⁺ imaging in the mouse cortex was performed with a custom-built two-photon microscope based on an open-source design (MIMMS 2.0, janelia.org/open-science) and controlled with Scanimage 5.7_R1 software (Vidrio Technologies) and National Instrument electronics. The Ti:Sapphire excitation laser (Tiberius, Thorlabs) was tuned to 930 nm and focused with a 16×0.8 NA objective (Nikon) below the cortical surface. The laser power (typically 25 mW measured at the objective) was modulated with Pockels Cells (350-80-LA-02, Conoptics) and calibrated with a Ge photodetector (DET50B2, Thorlabs). A 550 µm by 550 µm area of cortex was scanned at ≈30 frames/s using a resonant-galvo scanning system (CRS8K/6215H, CRS/671-HP Cambridge Technologies). Emitted fluorescence was detected with GaAsP photomultiplier tubes (PMT2101, Thorlabs) and the acquired 512 × 512 pixel images written in 16 bit format to disk. Behavioral event (trial start and stimulus onset) TTL pulses issued by the Bpod State Machine were received as auxiliary inputs to the Scanimage electronics and their timestamps saved in the headers of the acquired images. The timestamps were used to temporally align neuronal data to behavioral events.

We used a 2-photon imaging system described previously⁶³ . All imaging was conducted using a custom-built 2-photon 8 kHz resonant scanner using large aperture fluorescence collection optics (primary dichroic: 45.0 ×65.0 ×3.0 mm T865lpxrxt Chroma, US; secondary dichroic: 52.0 ×72.0 ×3.0 mm FF560-FDi02-t3 Semrock, US; green emission filter: 50.8 mm FF01-520/70 Semrock, US; red emission filter: 50.8 mm FF01-650/150 Semrock, US; GaAsP PMTs for green and red channels, PMT2101 Thorlabs, US) paired with a Nikon 16x water immersion, 0.8 NA, 3.0 mm working distance objective. Laser power was controlled with a Pockel cell (350-80LA modulator, 320RM 401 driver, Conoptics, US). Image acquisition was controlled through commercial software (ScanImage, Vidriotech, US). jRGECO1a expressing cells were imaged at 1020 nm (Chameleon Ultra II, Coherent, US). All frame scans lasted 1–2 minutes and were acquired at 120-240 Hz (40–60x optical zoom; 64–128 lines/frame, 64–128 pixels/line) from dendrites (oblique and tuft). Dendritic jRGECO1a signals were deconvolved to detect putative events by applying the OASIS software package^{64 (link),65 (link)} using an AR1 model with a pre-computed signal decay constant of 300 ms (Arg = 0.97; event threshold = 0.3). All dendritic calcium analyses were performed on the deconvolved events.
Reagent table can be found as Supplementary Table 1.