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Unexpanded and expanded specimens in 35 mm glass bottom dishes were fixed as described in the previous protocol. Wide-field fluorescent images were collected onto an EMCCD camera (Evolve 512, Delta Photometrics) with an oil-immersed objective (150 × /1.45, Olympus), yielding a pixel size of 106 nm. Emissions were collected through an objective and filtered by a 520 nm band-pass filter (FF01-520/35-25, Semrock) for Alexa488 or by a 655 nm band-pass filter (FF02-655/40-25, Semrock) for Alexa647. Airyscan imaging was performed using a commercial microscope (ZEISS, LSM880, Germany) with an additional Airy FAST detector module (Zeiss), equipped with a Plan-Apochromat 63 × /1.4NA oil objective (Zeiss; Plan-Apochromat 63 × /1.4 Oil DIC M27). Samples stained with BG-dsDNA-Alexa488 were excited using a 488 nm laser. Emissions were collected through a 495–550 nm band-pass filter, 570 nm long-pass filter, and a 1.25 airy unit (~60 μm) pinhole onto a 32 GaAsP detector element. Pictures were obtained using ZEN software (Zeiss; black edition) and pixel size was set to ~40 nm in the x and y directions. SIM imaging was performed using a commercial microscope (NIKON, A1&SIM&STORM, Japan) with a SIM model equipped with a 100 × /1.49NA oil objective (Nikon; CFI Apo TIRF 100 × /1.49 Oil), and the 488 laser was selected.

WFI was performed on day 1or 2 of the training step 3 (beginner) and on day 21 or 22 (expert) using a CMOS camera (FLASH4; C-13 440-20CU; Hamamatsu) system, adapted from a previous study (Kim et al., 2016 (link)). The cortical surface was imaged through a 2×/0.08 NA objective (PlanApo N; Olympus). For measurement of GCaMP6 emission, a 470 nm LED (Optogenetics-LED-Blue; Prizmatrix) was connected to the dichroic cube holder using a 1000-μm-diameter 0.50-NA fiber (M59L01; Thorlabs). The light was filtered with a 470 nm bandpass filter (FB470-10; Thorlabs) that was fiber-coupled into the dichroic mirror (FF495-Di03-25 × 36; Semrock). Fluorescence emitted from the cortical surface was passed through a 530 nm bandpass fluorescence emission filter (FF01-520/35-25; Semrock). Images were acquired using HCImage Live (Hamamatsu) at 20 Hz (50 ms/frame) and 192 × 192 pixels. Imaging was started by using the transistor-transistor logic (TTL) signal generated from the Arduino to synchronize the imaging and behavior data.

The NIR multimodal optical setup was described in our previous work^{14 (link),23 (link)}. It consists of a two-photon multimodal benchtop microscope (Leica SP8, Leica microsystems) able to perform four different optical imaging modalities: (1) One and two-photon fluorescence imaging (2) two-photon FLIM measurement, (3) SHG imaging and (4) one and two-photon Spectral imaging.
Analyses were conducted using the dedicated Leica software (LAS-X) as well as Matlab and imageJ. For FLIM measurements, 3 * 3 images mosaic per sample were analyzed using 810 and 890 nm the excitation wavelengths. The same technique was used also for 2PEF and spectral imaging, where 2PEF images were acquired using 890 nm and the spectral images using 810 nm. The collected fluorescence signal is divided into two detection channels. Each detection channel contain a super sensitive hybrid non descanned (HyD NDD) detector. In the first detection channel (channel 1), a bandpass filter with a bandwidth of 448 ± 20 nm (FF01-448/20-25, semrock, USA) was used to select the NADH fluorescence signal, and in the second detection channel (channel 2) another bandpass filter with a bandwidth of 520 ± 35 nm (FF01-520/35-25, Semrock, New York, USA) was used to select the FAD fluorescence signal.

This platform consists of a Leica TCS SP8 multimodal multiphoton fluorescence benchtop microscope (Leica Microsystems, Wetzlar, Germany) controlled via Leica’s acquisition software, where its technical details were published elsewhere (15 (link)). Briefly, this platform includes a Ti : Sapphire tunable laser source from 690 nm to 1,040 nm (Mai Tai DeepSee, Spectra-Physics). The objective used is a 25× water-immersion (HCX IRAPO L 25X NA 0.95, Leica). A hybrid internal detector (HyD, Leica, Germany) was used for spectral measurements, while two other external non-descanned hybrid detectors (HyD-RLD, Leica Microsystems, Wetzler, Germany) were used for two-photon fluorescence, SHG, and FLIM imaging. The first one is dedicated to select the nicotinamide adenine dinucleotide (NADH) fluorescence signal when using 800 nm as excitation wavelength and SHG signal when using 890 nm, using a 448 ± 20 nm band-pass filter (Semrock, FF01-448/20-25). The second one is dedicated to select the flavins (FAD) fluorescence signal using a 520 ± 30 nm band-pass filter (Semrock FF01-520/35-25). For fluorescence lifetime imaging acquisition, a time-correlated single-photon counting module (PicoQuant TCSPC module, Berlin, Germany) was coupled with the two external hybrid detectors, which permit performing NADH and FAD lifetime imaging at 800 nm.