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For multicolour luminescence imaging, five images were acquired with Semrock FF01-447/60 filter (for Nluc), Olympus BA460-510CFP (for CeNL), Semrock FF01-525/35 filter (for GeNL), Semrock FF01-562/40 (for OeNL) and Semrock FF01-593/40 (for ReNL). Five HeLa cells (expressing each fusion construct) were imaged with identical conditions to determine the coefficients for linear unmixing. The signals from NLuc, CeNL, GeNL, OeNL and ReNL were then separated by linear unmixing using these coefficients by PrizMage software (Molecular Devices).

HEK293 cells were cultured in a 35-mm coverslip bottom dish or a 12-well plate to obtain images and measure FRET efficiency. To obtain the image and FRET efficiency of a cell, we used an inverted microscope with a 60x oil objective lens and the three-cube FRET calculation^{61 (link),62 (link)} controlled by MetaMorph 7.6 (Molecular Devices, U.S.A). We mainly used three-cube FRET and mCherry (FF01-562/40, FF593-Di03, FF01-617/75, Semrock). The three-cube FRET efficiency (cube settings for CFP, YFP, and Raw FRET) was acquired from a pE-1 Main Unit to three-cube FRET (excitation, dichroic mirror, filter) through a fixed collimator: CFP (ET 435/20 nm, ET CFP/YFP/mCherry beam splitter, ET 470/24 nm, Chroma); YFP (ET 500/20 nm, ET CFP/YFP/mCherry beam splitter, ET 535/30 nm, Chroma); and CFP/YFP FRET (ET435/20 nm, ET CFP/YFP/mCherry beam splitter, ET535/30 nm, Chroma). The excitation LED and filter were sequentially rotated, the rotation period for each of the filter cubes was ~0.5 s, and all images (three for CFP/YFP/Raw FRET) were obtained within 2 s. Each of the images was acquired on a cooled 3 MHz (14 bit) EMCCD camera (iXon Ultra 888: ANDOR) with an exposure time of 100 ms with 1 × 1, 2 × 2, or 3 × 3 binning under the control of MetaMorph 7.6 software. Our FRET recording of the fluorophores was restricted in a range of CFP/YFP ratio from 0.5 to 2.0.

A FRET-based fluorescent ATP probe (GO-ATeam2) was used to measure intracellular cytosolic ATP levels^{38 (link)}. Briefly, the probe encodes FRET pair fluorescent proteins, green fluorescent protein (GFP), and orange fluorescent protein (OFP). Transfected neurons were visualized with an Olympus Fluoview (F1000) inverted microscope (Olympus Corp., USA) using the Plan Fluor ×63 (1.45 NA) oil-immersion objective lens (Olympus). The filters used for dual-emission ratio imaging of GO-ATeam were purchased from Semrock (Rochester, NY) and included an FF01-482/18 excitation filter, an FF495-DiO2 dichroic mirror, and two emission filters (FF01-520/35 for GFP and FF01-562/40 for OFP). The images were imported to ImageJ (NIH); CTCF values for each channel (GFP and OFP) were quantified and the ratio (OFP/GFP) was calculated using Microsoft Excel software.