Thorimagels software

Fluorescence signals were recorded using a two-photon microscope (ThorLabs Bergamo II, 12 kHz scanner) with a Nikon 16× water-immersion objective (NA 0.8) giving a field of view of 540 × 540 µm. The scope was operated using ThorImageLS software (v4.0.2019.8191, Thorlabs). In most experiments the microscope was rotated by 45° from vertical. Owing to physical constraints, pIC and S1 two-photon imaging was carried out in different animals and sessions. The excitation laser (InSight DeepSee, Spectra-Physics) was tuned to 940 nm, and the power never exceeded 100 mW. Emitted photons were bandpass filtered 525/50 (green) onto a GaAsP photomultiplier tube. Multi-plane (512 × 512 pixels) acquisition was controlled by a fast piezoelectric objective scanner, with planes spaced 45 μm apart in depth. Seven planes were acquired sequentially, and the scanning of the entire stack was repeated at about 5 Hz. Beam turnarounds at the edges of the image were blanked. Acquisition trials lasted 26 s and had an inter-trial interval of 30–60 s. Stimuli generation and hardware synchronization were carried out on a computer with a National Instrument card running custom-written Python code.

Fluorescence was monitored in L2/3 neurons in V1 at 920 nm using a commercial two-photon microscope (B-Scope, ThorLabs, Newton, New Jersey, USA) equipped with a tunable ultrafast laser (MaiTai BB DS-OL, Spectra-Physics, Santa Clara, USA) and a 16× water immersion objective (0.8 NA, Nikon, Tokyo, Japan). A 500–550 nm emission filter was used for dye detection. Scanning and image acquisition were controlled using ThorImageLS software (Thorlabs) at a sampling rate of 20.4 Hz. The size of the acquired images was 768 × 768 pixels from a field of view measuring 517.77 μm × 517.77 μm. The average power delivered to the brain was less than 70 mW. Every spontaneous recording lasted approximately 73.5 s, and each stimulus recording lasted 24.5 s. The focal plane and imaging position were checked and manually realigned with the initial image if necessary.