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After habituation and training, mice were used for Ca²⁺ imaging. Multiphoton fluorescence microscope (Femtonics, Budapest, Hungary) with a 16× NA 0.8 water-immersion objective (CFI75 LWD 16X W, Nikon, Tokyo, Japan) or with a 20× NA 0.8 water-immersion objective (XLUMPLFLN20XW, Olympus, Tokyo, Japan) was used for Ca²⁺ imaging in vivo. Ti:Sapphire laser (Chameleon Ultra, Coherent, USA) with an excitation wavelength of 920 nm was used to excite the fluorescence of GCaMP6f. Fluorescence signal from a brain area of 600 × 600 or 175 × 175 µm² (512 × 512 pixels) was recorded in resonant scanning mode at 30 frames/s. The signal was filtered with a 520/60 nm bandpass filter (Semrock, Rochester, NY, USA) and then detected with a GaAsP photomultiplier (H11706P-40, Hamamatsu, Japan). Simultaneously, autofluorescence was filtered with a 650/100 nm bandpass filter and detected with a second identical photomultiplier. Several imaging sessions of 10 min were carried out for each mouse. In parallel with Ca²⁺ imaging, the animal movements on the platform were monitored with locomotion-tracking software (Neurotar, Helsinki, Finland). All sessions were video recorded in infrared light. The experimental session did not exceed 2 h per mouse.

Two-photon calcium imaging of rd1-Thy1-GCaMP6s retina was carried out on a custom galvo-scanning microscope equipped with a 20×1.0 NA water immersion objective (XLUMPLFLN20XW, Olympus). Excitation at 920 nm was provided by a tunable Ti:Sapphire ultrafast laser (Chameleon Ultra, Coherent). Imaging parameters were controlled by ScanImage 3.8.1 software (http://scanimage.vidriotechnologies.com/): 256×256 pixels at 1.25 Hz (2ms per line). GCaMP6s emission was collected with a GaAsP PMT shielded by a longpass filter (ET500lp, Chroma).
Isolated retinas were cut into four-leaf clovers and transferred onto filter paper (0.45 mm nitrocellulose membranes, MF-Millipore) with the ganglion cell layer facing up. Oxygenated ACSF was then perfused over the retina at 34 °C for 30 minutes with a flowrate of 1 mL/min. An initial imaging session performed to account for potential two-photon sensitivity. Experimental imaging was performed with the laser power at the sample ≤5 mW. For pharmacological blockade of actional potentials, Lidocaine (2% in saline) was applied to the bath during corresponding recordings. Washout of Lidocaine was performed by continuously perfusing oxygenated ACSF at 34 °C over the course of two hours with a flowrate of 1 mL/min.

High-resolution two-photon excited fluorescence (TPEF) in vivo imaging was performed on a Prairie Ultima Multiphoton microscope equipped with a mode-locked Ti:sapphire laser (Chameleon Ultra II, Coherent, Santa Clara, California). Laser excitation was set at 910 nm for EGFP detection and a maximum power of $\sim 35\mathrm{mW}$ was used for data acquisition. Images were acquired with a water immersion lens (XLUMPLFLN20XW, Olympus, Tokyo, Japan; $20\times$ magnification, $\mathrm{NA}=1$ ) at $1024\times 1024\text{pixel}$ resolution and zoom = 1, leading to $600\mu \mathrm{m}$ field of view with linear resolution of $0.594\mu \mathrm{m}/\text{pixel}$ .

To obtain vascular and myocardial information on the mouse heart, the sample was placed on the HD-dfMOST system for sectioning and imaging. The whole sample was immersed in a water sink containing PI solution for real-time staining of the myocardial architecture. Whole-heart imaging was performed in a water bath. In our experiment, the sample was imaged at a voxel resolution of 0.32 × 0.32 × 1.0 μm³. The heart sample colabeled with myocardial architecture and blood vessels was imaged through two channels (GFP and PI, respectively).
The following devices were used in the HD-dfMOST system (Figure 1B): lasers (488 and 561 nm, Cobolt, Sweden), lenses (L1, AC050-008-A-ML, f 7.5 mm; L2, AC254-250-A, f 250 mm; L3, TL, AC254-125-A-ML, f 125 mm, Thorlabs, USA), a cylindrical lens (ACY254-100-A, f 100 mm, Thorlabs, USA), an objective lens (XLUMPLFLN 20XW, 1.0 NA, Olympus, Japan), dichroic mirrors (ZT488/561rpc Chroma USA), excitation filters (ZET488/561 m, Chroma, USA), sCOMS cameras (ORCA-flash4.0 V3, Hamamatsu, Japan), and an XYZ linear stage (x-axis XML210, y-axis XMS100, z-axis GTS30V, Newport, USA).