Fb470 10

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.

As easily accessible common light sources for optical excitation, we used ultraviolet, blue, and green light-emitting diodes (LEDs) with emission wavelengths of 415 nm (FWHM = 14 nm), 470 nm (FWHM  =  25  nm), and 530 nm (FWHM = 33 nm) purchased from Thorlabs Inc. (Newton, NJ, USA). Bandpass filters of 410, 470, and 530 nm (FB410-10, FB470-10, and FB530-10; Thorlabs Inc.) were placed between the light source and the PUF device. The optical power was kept at 1, 3, and 10 μW mm⁻² for 415-nm, 470-nm, and 530-nm LEDs at the surface of PUFs. To image the PUFs, we used a charge-coupled device (CCD) camera (Princeton Instruments PIXIS 1024B, Trenton, NJ, USA) with a conventional zoom lens (MVL7000, Navitar, Rochester, NY, USA) via a liquid crystal tunable filter with a FWHM of 7 nm (VariSpec VIS-07-20; PerkinElmer, Inc., Waltham, MA, USA). As a result, the following set of excitation and emission bands was selected such that λ_ex = 415 nm and λ_em = 460 nm; λ_ex = 470 and λ_em = 510 nm; λ_ex = 470 and λ_em = 560 nm; λ_ex = 530 and λ_em = 630 nm, optimized for eCFP, eGFP, eYFP, and mKate silk, respectively.

Photometry was performed as described in detail previously^{54 (link)}. Briefly, we used a 405-nm LED and a 470-nm LED (Thorlabs, M405F1 and M470F1) for the Ca²⁺-dependent and Ca²⁺-independent isosbestic control measurements. The two LEDs were band-pass filtered (Thorlabs, FB410-10 and FB470-10) and then combined with a 425-nm long-pass dichroic mirror (Thorlabs, DMLP425R) and coupled into the microscope using a 495-nm long-pass dichroic mirror (Semrock, FF495-Di02-25 3 36). Mice were connected with a branched patch cord (400 mm, Doric Lenses, Quebec, Canada) using a zirconia sleeve to the optical system. The signal was captured at 20 Hz (alternating 405-nm LED and 470-nm LED). To correct for signal artifacts of a non-biological origin (i.e., photo-bleaching and movement artifacts), custom Matlab scripts leveraged the reference signal (405-nm), unaffected by calcium saturation, to isolate and remove these effects from the calcium signal (470-nm). Correlation of df/F activity and threat proximity during EPM test and Rat assay were calculated as previously described^{26 (link)}.

To measure photoswitching rates ± calcium, purified rsCaMPARI in low (10 mM EGTA) or high (10 mM CaEGTA) calcium conditions buffered with 25 mM Tris, 100 mM KCl, pH 7.5 was illuminated with a 470 nm LED (Mightex) with different bandpass filters (parts: FB450-10, FB460-10, FB470-10, FB480-10, FB490-10, and FB500-10, Thorlabs; ET485/25x, Chroma). Output spectra were measured with a USB4000-UV-VIS Ocean Optics spectrometer. Light intensities were measured on a power meter (Coherent #1098580) using a silicon photodiode (Coherent #1098313). Green fluorescence was measured on a plate reader (Tecan) at various timepoints and a single exponential rate was fitted (Prism, Graphpad) to the change in green fluorescence. The optimal wavelengths for rsCaMPARI off-switching (470–490 nm) are defined as ‘marking light’ and the optimal wavelengths for rsCaMPARI on-switching (390–410 nm) are defined as ‘erasing light’.