Fb410 10

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)}.