The unique absorption properties of graphene enable a long carrier lifetime of charge carriers contributing to the carrier-multiplication due to impact ionization and, hence, large photocurrent and high quantum efficiency. The proposed p+-BLG/n-Hg0.8133Cd0.1867Te photodetector is characterized and analyzed in terms of different electrical and optical characteristic parameters using computer simulations. The obtained results are further validated by developing an analytical model based on drift-diffusion, tunneling and Chu's methods. The photodetector has demonstrated a superior performance including improved dark current density (∼1.75 × 10−14 µA cm−2), photocurrent density (∼8.33 µA cm−2), internal quantum efficiency (QEint ∼ 99.49%), external quantum efficiency (QEext ∼ 89%), internal photocurrent responsivity (∼13.26 A W−1), external photocurrent responsivity (∼9.1 A W−1), noise equivalent power (∼8.3 × 10−18 W), total noise current (∼1.06 fA), signal to noise ratio (∼156.18 dB), 3 dB cut-off frequency (∼36.16 GHz), and response time of 9.4 ps at 77 K. Furthermore, the effects of different external biasing, light power intensity, and temperature are evaluated, suggesting a high QEext of 3337.70% with a bias of −0.5 V near room temperature.We present a high-performance bilayer graphene (BLG) and mercury cadmium telluride (Hg x1−Cdx=0.1867Te) heterojunction based very long wavelength infrared (VLWIR) conductive photodetector.We present a high-performance bilayer graphene (BLG) and mercury cadmium telluride (Hg The IR detection region is categorized into short wavelength IR (SWIR: 1–3 µm), mid wavelength IR (MWIR: 3–5 µm), long wavelength IR (LWIR: 8–14 µm) and very long wavelength IR (VLWIR: 14–30 µm) ranges.12 The potential applications of IR photodetectors include biomedical and thermal imaging, gas sensing, night vision, spectroscopy, and free space communication etc.4,13–15 Rapid advances are being made in developing inexpensive narrow bandgap semiconductor photodetectors with improved sensitivity and longer wavelengths. As a result, low dimensional structures, including quantum-well, quantum-dot, and quantum-wire, based IR photodetectors are reported with improved performances, but with expensive fabrication processes.16–19 On the other hand, the low leakage current, tunable bandgap, better stability, low thermal generation rate and relatively high absorption coefficient make the ternary alloy mercury cadmium telluride (MCT: Hgx1−CdxTe) a suitable material for high performance IR detectors.12,14,20–22 So far, numerous Hgx1−CdxTe-based IR photodetectors with different configurations such as p–n,23,24 p–i–n,14,25 dual band IR detector,15,26 and avalanche photodiode27 have been reported at cryogenic temperatures. The high dark current limited by Auger recombination processes and low temperature operations are the major disadvantage of MCT based IR photodetector.14,20,28,29 Hence, there is a requirement to design and develop MCT based IR photodetectors demonstrating higher efficiencies near room temperature.Infrared (IR) photodetectors have been successfully demonstrated from a wide variety of narrow bandgap semiconductors (Si and Ge)30 The Gr/semiconductor heterojunction based photodetectors are expected to demonstrate a low dark current, low power dissipation, small parasitics, higher breakdown voltage, and high response speed than that of conventional homostructures. The Gr has been successfully composited with different materials like ZnO,31,32 Si,33 CdS34 (for UV applications), CdSe,35 GaN36 (for visible applications), and Si,1,37 Ge,3 PbS,38 GaAs39 (for IR applications). However, the zero bandgap and small optical absorption (∼2.3%) of monolayer graphene (MLG) result in limited photocurrent responsivity (<1 A W−1) and photocurrent (Ilight) to dark current (Idark) ratios (Ilight/Idark).6,30,33,40–42 Accordingly, several efforts including utilisation of few layer graphene, Gr quantum dots, inducing small bandgap in Gr layers using either doping or under transverse electric fields are made to improve the charge injection and separation, suppressing dark current with improved efficiency.11,13,43–46 The intentional doping in monolayer and bilayer Gr shifts the Fermi-level either upward or downward, creating a bandgap opening.47 To the best of our knowledge, a maximum bandgap of 430 meV is found in p-doped bilayer graphene (BLG).48 Xu et al.49 demonstrated 25 times higher electrical conductance and optical transmittance of 80% from few layer (5–10) graphene transferred onto MCT substrate in the MWIR spectral region at 77 and 300 K. Despite such studies, no photodetector combining BLG with MCT is successfully studied yet. Hence, here we propose and simulate a BLG/MCT based photodetector providing excellent light absorption and electron transport. The device exhibits a low dark current and high temperature operation due to lower thermo-generation rate. In this work, we investigate the effect of different external bias, incident light power intensity and temperature on the performance of proposed heterojunction photodetector. It is evident from the results that high external quantum efficiency (QEext) (>100%) is achieved due to long lifetime of photo-induced hot carriers in the VLWIR spectral region. The obtained results are further validated by developing an empirical model based on drift-diffusion, tunneling and Chu's methods in VLWIR, suggesting potential applications in next-generation high-performance, ultra-low-power, and cost-effective IR photodetectors for optoelectronics devices.The excellent electrical and optical properties of graphene (Gr) enables utilisation of Gr as a transparent electrode integrated with conventional photodetectors for ultraviolet (UV) to IR regions.x1−Cdx=0.1867Te based VLWIR photodetector. The highly doped p+-type BLG conformably cover 10.0 µm wide lightly doped n-type Hg0.8133Cd0.1867Te to form a heterojunction photodetector. The p+-doping of the Gr is typically being achieved through the chemical doping with HNO3,50,51 FeCl3,52 AuCl3,53,54 SOCl2,55,56 and NbCl5  are made as anode and cathode to collect the photo-generated charge carriers. The nominal thickness of 2 nm for BLG is considered for our studies which is in consonance with previously reported values.59–61 The proposed p+-BLG/n-Hg0.8133Cd0.1867Te photodetector can be grown on lattice matched CdZnTe substrate. CdZnTe substrate is chosen because MCT/CdZnTe interface offers less interface trap charge as compared to Si, Ge, and CdTe substrates.24 In this paper, we have not considered the substrate effect, as it does not affect the device performance significantly. The Silvaco Atlas software is utilised to design and evaluate the electrical and optical performances of BLG/HgCdTe photodetector. The VLWIR radiations are incident from the p+-BLG cladding window over the narrow bandgap n-Hg0.8133Cd0.1867Te active/absorbing layer. The p+-BLG is used as a light absorber. The lightly doped n-Hg0.8133Cd0.1867Te generates total dark current and photocurrent. The bandgap and doping concentration of active region are selected to give better absorption of VLWIR radiations suppressing thermally generated carriers. In order to simulate the device, a bandgap of 250 meV for BLG is considered.62,63 When VLWIR light illuminates the p+-BLG/n-Hg0.8133Cd0.1867Te heterojunction, the hot photo-carriers transport from the active layer to the Gr layer due to built-in electric field (Ebuilt-in).64 As a result, the change in graphene conduction is observed according to the relation, Δσ = Δnqµ,65,66 where Δn represents variation in carrier-concentration of graphene; q corresponds to the electronic charge; and µ is the carrier mobility. The schematic illustration of depletion region with a barrier height (ϕB) between the BLG and HgCdTe is given in χ and electrostatic potential ∇V. The electrons are dominated charge carriers than holes. The electrons travel from n-region into the lower energy states in p+-region, leaving the positively charged empty states in the n-region. Accordingly, the energy band bends upward in the vicinity of the p+–n heterojunction to form an Ebuilt-in. The doping profile of the material determines the barrier height, however, potential variation results in energy band drop at p+–n interface junction23 as shown in 67 It is also observed from 68 The absorbed photo carriers undergo hot carrier multiplication (CM) by impact ionization 69–72 in p+-BLG, subsequently cross the barrier through internal photoemission (IPE) process ,33 and finally be collected by n-Hg0.8133Cd0.1867Te contributing the total photocurrent. The carrier multiplication factor can further be tuned with chemical doping.73,74 The fast recombination rate (in picosecond) of the photogenerated carriers results in a maximum multiplication factor of 4.3 by the impact ionization process.75 Thus carrier multiplication, i.e. internal gain mechanism in BLG will enhance the photocurrent Ilight of the device due to the increase in carriers.76 The increase in photocurrent will result in higher internal quantum efficiency (QEint),33 facilitates to break through the upper-limit of traditional MCT based IR photodetectors. This indicate that our BLG composite photodetectors can still work effectively under a low illumination conditions. In our device, we have achieved a carrier multiplication factor of 1.25 for p+-doped graphene at 250 K, which is in good agreement to the earlier reported values.73,74 In addition, an equivalent Mixed-Mode circuit is utilised for estimating device performances in dark condition as shown in Rsh, junction capacitance Cj, and load resistance RL with series resistance Rs at an applied bias V for generating carriers contributing to the device current. The effect of illumination is implemented by a constant current source Ilight in Silvaco Mixed-Mode simulator.The inset of nd NbCl5  utilisinw = [2ε0εr1εr2(Vbi − V)(NA2 + ND2)/(q(εr1NA + εr2ND)NAND)]1/2 of the heterojunction is found to be 64.28 nm for a built-in potential (Vbi = Vip + Vin) of 21 mV. Here, Vip and Vin are the barriers corresponding to energy band bending at p+- and n-regions, respectively. ε0 is the absolute permittivity and εr1 & εr2 represent the relative permittivity in p+- and n-regions, respectively. NA (2 × 1022 cm−3) and ND (1 × 1016 cm−3) corresponds to the acceptor and donor doping concentrations in p+- and n-regions, respectively. The thickness of bilayer graphene is considered to be 2.0 nm, which is considerably smaller than that of MCT (10.0 µm). Accordingly, the depletion region extends inside the MCT (n-side) resulting in gradual shrinkage in band diagram towards BLG/MCT interface as observed in Cj = ε0εr2A/w in self-powered mode is found to be 0.98 fF. The device exhibits the transit time τtr = w/vsatn of 6.4 ps, where vsatn is the carrier saturation velocity (106 cm s−1) in n-region. The dependence of εr2 on x composition for n-region is approximated by:12In self-powered mode, the depletion layer width +-BLG/n-Hg0.8133Cd0.1867Te photodetector is electrically characterized by solving continuity, carrier transport and Poisson equations with optimized boundary conditions as approximated by Boltzmann's transport model.15 Here, BLG is considered as 3D in nature analogous to earlier reported studies demonstrating the integration of multilayer graphene with other conventional semiconductors which couples 2D transport equations to 3D equations.67 A 2D semiconductor material has one physical dimension of the order of Fermi wavelength λF. In our case,  of graphene is estimated to be around 0.56 nm for the sheet carrier density (n) of 4 × 1015 cm−2. The obtained value of λF is smaller than the considered thickness of doped BLG (2 nm), hence, it is necessary to consider 3D equations for estimating device characteristics. Further, to reproduce the carrier transport in graphene a drift-diffusion approach is implemented to degenerate semiconductor and parabolic shape of the conduction band.77 The Newton–Richardson iteration method and concentration dependent Analytic model are used to estimate the carrier mobility in the photodetector.15,24 In order to characterize the carrier lifetime and the dark current in the proposed photodetector, Shockley–Read–Hall (SRH), Auger, optical (band-to-band), trap assisted tunneling (TAT), and band-to-band (BTB) standard tunneling mechanism models are considered. The doping and carrier densities are evaluated using Fermi–Dirac statistics.15,24,78The px of cadmium in the Hgx1−CdxTe tunes the energy bandgap in VLWIR with a cut-off wavelength of 20.6 µm. For n-Hgx1−CdxTe, the energy bandgap (Egn), electron affinity (χn), and intrinsic carrier concentration (nin) as a function of x composition and lattice temperature T are approximated by 12,24,79k is the Boltzmann constant.The performance of the photodetector at 77 K is evaluated analytically for the operation at the wavelength of 20.6 µm. The photons having energy greater than the bandgap create electron–hole pairs in the lightly doped n-region. The mole fraction  of n-Hgx1−CdxTe material is estimated by using Weiler's expression80 (eqn S1 and S2NCB) and holes (NVB) in conduction and valence bands, respectively, intrinsic carrier concentration (ni), and carrier (electron and hole) lifetimes are listed in 12,24,67,81+–n heterojunction VLWIR photodetector for different biasing conditions. The maximum value of electric field (Emax) is found to be 17.8, 45.4, and 64.6 kV cm−1 at the heterojunction of the device with an external bias of 0, −0.5, and −1 V, respectively. The electric field E(x) across the device can be written as:x+p and xn are the depletion width in p+- and n-regions, respectively.The electron effective mass x+p either almost fully depleted or very thin (≈3.21 × 10−16 m in our case), hence, can be safely neglected. The electron–hole pairs from MCT can be effectively separated by high built-in Ebuilt-in to generate the photocurrent. The dark current density (Jdark) is a combination of diffusion current density (JDIFF) in the neutral p+- and n-regions, drift current density (JGR) due to generation–recombination of charge carriers in the depletion region and the tunneling current density (JTUN). JTUN consists of the contribution of both the trap-assisted tunneling (TAT) and band-to-band (BTB) tunneling. Accordingly, the total dark current density as a function of voltage and ambient temperature is given by:BLG is atomically thin with a thickness of the order of nm. This may create the depletion region width 24V represents the applied bias voltage, the diffusion component of current density for holes (Jp)n and electrons (Jn)p in n- and p+-regions, respectively, are given as:nin and nip are the intrinsic carrier concentrations in n- and p+-regions, respectively. µp and µn are the hole and electron mobilities, respectively. τp and τn represent the hole and electron lifetimes, respectively. γp = SpLp/Dp and γn = SnLn/Dn are the ratios of surface to bulk recombination velocities in p+- and n-regions, respectively. Here, Sp = 103 cm s−1 and Sn = 105 cm s−1 are the surface recombination velocities of holes and electrons, respectively at the heterojunction.24 and  are the diffusion lengths of holes and electrons. Dp = µpkT/q and Dn = µnkT/q represent the diffusion coefficients of holes and electrons, respectively. The valence and conduction band-edge discontinuities, after the formation of the p+–n heterojunction are given by ΔEVB (=ΔEg − ΔECB) and ΔECB (=χn − χp). χn and χp are the electron affinities of the wide and narrow bandgap materials, where χn > χp. ΔEg (=Egp − Egn) is total bandgap discontinuity and Egp and Egn represent the energy bandgaps in the p+- and n-regions.The total diffusion current density is evaluated as:+–n heterojunction is strongly affected by the trap levels at the heterojunction particularly in depletion region. The electron and the hole components of current density due to the generation–recombination in the depletion region is given as:82τSRH = 1/σNfvth is SRH generation–recombination lifetime of carriers. σ = 6.9591 × 10−16 cm2 represents the capture cross section of minority carriers. Nf is the SRH trap density.  is the thermal velocity of minority carriers.The transfer of charge carriers across the pvia a trap level within the bandgap. These trap states are the intermediate energy levels generated due to the existence of impurities in the material. The TAT component of current density is evaluated as:82Nt is the trap density different from SRH trap density, and h is the Planck's constant. Wc and P represent matrix elements associated with the potential and interband matrix elements, respectively. The position of the trap levels in the bandgap is indicated by Et.The current related to TAT corresponds to the tunneling of electrons to the conduction band 82ℏ = h/2π is the reduced Planck's constant, E represents the electric field across the depletion region.The high reverse bias causes bending in energy band to tunnel the electrons from valence band to the conduction band as shown in J) as function of applied voltage varying from −1.0 to 0.3 V. The results are obtained under dark condition, and at a radiation of incident power intensity (Pin) of 1 W cm−2 at 20.6 µm. For the Mixed-Mode simulations of the circuit shown in Ilight, Cj, and Rsh are obtained at zero-bias condition. The proposed photodetector exhibits a low dark current density of 1.75 × 10−14 µA cm−2 and a photocurrent density of 8.33 µA cm−2 at zero-bias (self-powered mode) which in turn improves the signal-to-noise ratio (SNR), and hence specific detectivity. The simulated values of Jdark–V characteristic are well in accordance with the values obtained from the analytical modeling. The obtained Jdark is smaller than that of the state-of-art photodetectors such as Hgx1−CdxTe based IR photodetector,24 Gr-Silicon Schottky IR photodetector,1 Gr-Germanium Schottky IR photodetector,3 Gr-GaAs IR photodetector39 and armchair graphene nanoribbons IR photodetector.83Jlight–V characteristics under illumination at 20.6 µm for different Pin varying from 1 mW cm−2 to 2 W cm−2 are shown in Jlight increases with the incident power, and shifts towards the positive voltage region due to unidirectional charge transport mechanism. The two important figure-of-merits to evaluate the electrical performance of photodetectors are Jlight/Jdark ratio and linear dynamic range33 (LDR (dB) = 20 log(Jlight/Jdark)). Jlight, Jlight/Jdark ratio, and LDR for various Pin values at zero-bias and 77 K. The device exhibits Jlight/Jdark ratio of 4.8 × 1014 and LDR of 293.55 dB at 2 W cm−2 in self-powered mode and are better than that of Gr–Si based heterojunction photodetector.84,85 The photocurrent density Jlight and incident power Pin satisfy the relationship86Jlight ∝ Pinα, where α is an empirical value related to the recombination process of the photoexcited carriers. From α = 1.0 is obtained for reverse biases varying from −1.0 to 0.0 V. The linear response of photocurrent density as a function of incident power indicates that the recombination loss is negligible for the proposed heterojunction photodetector. NET with respect to temperature variation at a bias of −1.0 V and incident power of 1 W cm−2. The increase in temperature decreases the resistance and LDR, whereas, increases the current density. The (RA)NET arises due to different current densities, i.e., JDIFF, JGR, JTAT and JBTB, and can be written as:The  of 0.3 MΩ cm2 in self-powered mode which in turn results in high specific detectivity.The simulated and analytical resistance area product of the proposed photodetector for different voltages varying from −1.0 to 0.3 V at 77 K is shown in x1−CdxTe material within the Kane region is calculated by Chu's empirical relation.87,88 For the photon energy Ep < Egn (tail region) and Ep > Egn (Kane region), the absorption coefficient is approximated by:α0 = ex) dependent complex refractive index for Gr is calculated by42nGr(λ) = 3.0 + i1.8153λ, whereas, absorption coefficient is approximated by α+p(λ) = 1.8153 × 4π/λ. The complex refractive index of MCT as a function of x composition and temperature is given as:89In order to compute the optical characteristics, complex refractive indices are described for both the BLG and MCT. The wavelength (24K = λαn(λ)/4π.The imaginary part of the refractive index for MCT depends on the wavelength and absorption as.Ri), specific detectivity (D*), and noise equivalent power (NEP) of proposed photodetector are evaluated using computer simulations. The two types of quantum efficiencies, i.e. internal QE (QEint) and external QE (QEext) are often considered for a photodetector. The QEint determines the internal photocurrent responsivity (Rinti), whereas QEext gives the external photocurrent responsivity (Rexti). The QEext, Rexti, D*, and NEP of the device are further evaluated using the analytical model. The QEext measures the wavelength dependent gain of the photodetector and can be given as3269–74 which is a function of bias voltage and ambient temperature, c is the speed of light, Jλ is the photocurrent density, and λ represents the wavelength of the incident radiation.The spectral response of the proposed photodetector as a function of wavelength is shown in Fig. S1.ext of the photodetector comprises of neutral p+-(QEext)+p, neutral n-(QEext)n, and the depletion (QEext)dep regions, and can be written as:24,90–92R+p and Rn are the Fresnel reflection coefficients at the entrance and p+–n interfaces, respectively.The net QEint of the IPE process is the number of carriers emitted to HgCdTe per absorbed photon, and QEext is the number of carriers emitted to HgCdTe per incident photon. The Rinti, Rexti, D*, and NEP are calculated using following relations:3,24,32,33,93f and A are the bandwidth, and active area, respectively. Here, NEP is estimated at a unity bandwidth, i.e. Δf = 1 Hz.The photocurrent responsivity is a ratio of photocurrent to the incident light power, whereas, quantum efficiency measures the sensitivity of the photodetector. On the other hand, the specific detectivity describes the smallest signal that a photodetector can detect, however, noise equivalent power is the minimum incident signal that a photodetector can resolve from the noise. The QEext, Rexti, and NEP for the proposed photodetector as a function of wavelength at a of −0.5 V and a radiation of 1 W cm−2 at 20.6 µm and 77 K. The device exhibits a high QEint ∼ 99.49% ), maximum external photocurrent responsivity, Rexti(max) and minimum NEP (NEP(min)) under IR illumination are shown in ext(max), and Rexti(max) resulting in a lower NEP. The increase in both QEext(max), and Rexti(max) with the reverse bias voltage is mainly due to the increased drift velocity of the photo-induced charge carriers along with the increased probability of exciton separation and acceleration with the bias voltage, which eventually contribute to the total photocurrent in the external circuit. The increase in QEext is attributed to the carriers passing thin depletion region multiple times at a certain reverse bias.32Rexti due to the increase in photocurrent which decreases both QEext and NEP. The decrease in QEext with incident power could also be attributed due to the self-induced-heating increase in the carrier scattering and hence the rate of charge carrier recombination. Furthermore, the increase in reverse bias and incident power results in improved D* as shown in Fig. S2b and c,The influence of reverse bias voltage, incident power intensity, and temperature on the maximum external quantum efficiency (QEext(max) increases from 10.68 to 3337.70%, whereas the Rexti(max) increases from 0.51 to 62.36 A/W for the temperatures varying from 30 to 250 K. The maximum D*  changes from 3.8 × 1015 to 8.08 × 1012 cm Hz1/2 W−1 for temperatures 30–180 K, and from 3.04 × 1011 to 1.29 × 1013 cm Hz1/2 W−1 for the temperature range 200–250 K  varies from 1.66 × 10−19 to 4.90 × 10−17 W for the temperature range 30–250 K as shown in ext(max), Rexti(max),  and NEP(min) of 3337.70%, 62.36 A W−1, 1.29 × 1013 cm Hz1/2 W−1 and 4.90 × 10−17 W, respectively, at near room temperature.The effect of temperature variation on optical parameters of the device is shown in ext more than 100% are generally associated with the well-known carrier multiplication effect in graphene.69–74 Such multiple hot-carrier generation can be explained with the schematic representation of energy band diagram of proposed device as shown in 74 Moreover, under the illumination of high optical energy, light can also be absorbed in both the M- and K-points of the Brillouin zone in graphene resulting in enhanced carrier multiplication effect.33The high values of QEext.The combined effect of carrier multiplication (in graphene) and presence of high electric field across the junction causes transport of multiple electrons (holes) to the positive (negative) bias electrodes which finally contributes to the net enhanced photocurrent and QEV,T) can be estimated from the simulated J–V characteristics under dark and illumination conditions at different temperatures as:27The CM and Johnson–Nyquist (ij) noise currents are calculated by82 and , where Idark is the dark current RNET = (dI/dV)−1, is the net resistance of photodetector in dark condition at reverse bias and Δf = 1 Hz is the bandwidth. The total noise current (in)93 and SNR82 are given by in = is + ij and In addition, we have estimated the shot (quantum) and Johnson–Nyquist  noise,in and SNR are 1.06 fA and 156.18 dB, respectively, for −0.5 V bias and a radiation of 1 W cm−2 at 20.6 µm and 77 K. f3 dB) and response time (tr) as a function of temperature at −0.5 V bias with incident power of 1 W cm−2. Beyond 3 dB cut-off frequency, the current/gain starts rolling-off quickly. The response time suggests the increase in output signal from 10–90% of the maximum value and is related to 3 dB cut-off as85The estimated values of tr decreases from 87.4 to 2.6 ps as shown in 82 It can be further increased with the increase in reverse bias voltage and reduction in the device active area inducing reduced capacitance. To the best of our knowledge, the obtained results for the proposed photodetector are better than that of previously reported graphene based photodetectors.1,3,29,30,33,37,39,43,82–85,93–103The results suggest exponential increase in 3 dB cut-off frequency from 3.89 to 129 GHz due to the existence of high saturation velocity and high carrier mobility, whereas +-BLG and few layer graphene/n-Hg0.8133Cd0.1867Te heterojunction VLWIR photodetector. The various electrical and optical characteristic parameters are computed and analysed using computer simulations and are further validated by an analytical model based on drift-diffusion, tunneling and Chu's methods. The dark current density is reduced due to lower thermo-generation rate of BLG and MCT heterostructure, offering the high quantum efficiency, high photocurrent responsivity, high specific detectivity, low noise equivalent power, low noise current, and better signal to noise ratio. The near room temperature external quantum efficiency of 3337.70% and responsivity of 62.36 A W−1 at −0.5 V bias are achieved in addition to a higher 3 dB cut-off frequency (∼129 GHz) and short response time (∼2.6 ps). Such superior performances are obtained due to hot carrier multiplication mechanism in graphene. The obtained results suggest the utilisation of p+-BLG/n-Hg0.8133Cd0.1867Te heterostructure for next-generation high-performance, ultra-low-power, low noise, and cost effective IR photodetection.In summary, we have demonstrated a high-performance pThere are no conflicts to declare.RA-008-C8RA07683A-s001


The purpose of the study is to explore the mechanism of NRAGE enhancing radioresistance of esophageal squamous cell carcinoma (ESCC) in 2D and 3D levels.Stably NRAGE-overexpressed ESCC cells and 3D-printing models for ESCC cells were established. Then, cellular malignancy indexes, such as cell morphology, proliferation, radioresistance, motility, apoptosis, cell cycle, and proteins of the Wnt/β-catenin pathway, were compared between radioresistant and its parental cells in 2D and 3D levels. Additionally, 44 paraffin ESCC specimens with radical radiotherapy were selected to examine NRAGE and β-catenin protein expression and analyze the clinical correlation.Experiments in 2D culture showed that morphology of the Eca109/NRAGE cells was more irregular, elongated spindle-shaped and disappeared polarity. It obtained faster growth ability, stronger resistance to irradiation, enhanced motility, reduced apoptosis ratio and cell cycle rearrangement. Moreover, Western blot results showed β-catenin, p-Gsk-3β and CyclinD1 expressions were induced, while p-β-catenin and Gsk-3β expressions decreased in Eca109/NRAGE cells. Experiments in the 3D-printing model showed Eca109/NRAGE cell-laden 3D scaffolds had the advantage on growth and spheroiding according to the brightfield observation, scanning electron microscopy and Ki-67 IHC staining, and higher expression at the β-catenin protein. Clinical analysis showed that NRAGE expression was higher in tumor tissues than in control tissues of ESCC patients from the Public DataBase. Compared with radiotherapy effective group, both NRAGE total and nuclear and β-catenin nuclear expressions were significantly upregulated from ESCC specimens in invalid group. Further analysis showed a positive and linear correlation between NRAGE nuclear and β-catenin nuclear expressions. Additionally, results from univariate and multivariate analyses revealed NRAGE nuclear expression could serve as a risk factor for ESCC patients receiving radical radiotherapy.ESCC cells with NRAGE nuclear accumulation demonstrated greater radioresistance, which may be related to the activation of the Wnt/β-catenin signaling pathway. It indicated that NRAGE nuclear expression was a potential biomarker for monitoring radiotherapeutic response. Esophageal cancer (EC), arising from esophageal epithelial cells, is an epidemic malignancy with conspicuous geographic distribution worldwide. It is fairly well known that China is one of the regions with a high incidence rate of esophageal squamous cell carcinoma (ESCC), which has an enormous burden and is a major histological subtype accounting for 95% of ECs in China –4. Stativia binding to p75 neurotrophin receptor (P75NTR) (via its interaction with apoptosis antagonizing transcription factor (AATF). Zou et al.  , 17, Che(P75NTR) , XIAP-TA(P75NTR) , and UNC(P75NTR)  and inhi(P75NTR)  and angi(P75NTR) . Contrad(P75NTR) , 23–26. (P75NTR)  found thu et al.  proved tin vitro system and more short research cycles than the 2D in vivo system. This study aimed to confirm the tumor promotor function of NRAGE in ESCC and mechanism that it can accelerate cell growth and survival and induce radioresistance in 2D and 3D culture levels and confer poor prognosis in the clinical setting.Originally, our previous studies indicated that NRAGE was significantly overexpressed in the nucleus of ESCC cells with radioresistance  and knocAll 44 patients who were clinically and histopathologically diagnosed with primary ESCC based on the WHO criteria, received radical RT through conventional fractionated RT by 6MV X-ray linear accelerator at the Second Hospital of Hebei Medical University from January 2010 to December 2015. The curative effects in the 44 patients were determined 1–3 months later after RT referred to the evaluation standard of esophageal barium swallow. The tissue specimens of the patients were collected, fixed in 4% formalin, and embedded in paraffin. The informed consent of the patients was obtained, and the study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (No. 20160275). Patient baseline characteristics are shown in ® WS, Hangzhou, China) was sterilized by 75% v/v ethanol and irradiated under UV for 30 min. E and E/N cells (5 × 106) were suspended in 0.2 ml culture medium, followed by the addition of 3 ml of a gelatin–sodium alginate blend . Gelatin and sodium alginate were purchased from Sigma . The temperature of 3D-printed platform was set at 8°C measuring 10 × 10 × 1.4 mm in size. After printing, the hydrogels were soaked in 3% calcium chloride for 3 min for a crosslink reaction. Subsequently, the 3D bioprinted scaffolds were incubated at 37°C with 5% CO2.Before printing, the 3D printed workstation  according to the instructions of the manufacturer. 3D cell-laden constructs were immersed in 1 ml PBS containing 8 μM PI  and 2 μM calcein AM  under the conditions of protection from light at room temperature reaction for 15 min and then washed with PBS. Stained cells were imaged using an inverted fluorescence phase contrast microscope . Live/dead cells were counted in five random fields at ×100 magnification for each sample. The cell death rate was calculated as follows: ratio of cell survival = number of living cells/(number living cells + dead cells) × 100%.control − A’570 control)/(A600control − A’600 control); A570 control and A600control, the OD values at 570 and 600 nm of cell-free alamarBlue working solution; A’570 control and A’600control, the OD values at 570 and 600 nm of cell-free medium.E and E/N cell proliferation in 3D bioprinted hydrogels were tested using an alamarBlue™ cell viability reagent . 3D-printed scaffolds were washed with PBS, and 500 μl of alamarBlue working solution  was added to each well of a 24-well plate. Then, the 24-well plate was incubated at 37°C and 5% CO2 for 2 h. Then, each 100 μl of working solution was transferred to a 96-well plate, and the absorbances at electron fixation solution at 570 and 600 nm on a multi-function microporous plate detector . The scaffolds were cultured for 19–21 days, and the OD values at 570 and 600 nm were detected every 2 days. The cell proliferation ratio calculation was as follows: reduction rate (%) = (A570 − A600 × R) × 100%; R =  for the night at room temperature and washed three times with PBS for 15 min. The samples were soaked in a series of ethanol solutions  for 15 min in each solution for dehydration. Subsequently, the scaffolds were dried in the ventilated kitchen. Then, the constructs were coated with platinum (5 nm thickness) and imaged with an Ultra-55 SEM .Experimental analyses were conducted as described previously , 27 and In vitro experiments were analyzed by unpaired two-sided Student’s t-test, Welch’s t-test, one-way analysis of variance (ANOVA) or two-way ANOVA. The total and nuclear protein expression of NRAGE and β-catenin in ESCC tissues were analyzed by Mann–Whitney U test. Survival curves were plotted using the Kaplan–Meier method and compared using the log-rank (Mantel–Cox) test. These statistical analyses were conducted by GraphPad Prism 8.0 software. Clinicopathological characteristics of patients with EC following radical RT were analyzed using the chi-squared test. The correlation between NRAGE or β-catenin and clinicopathological features of patients was analyzed using Spearman analysis. The linear correlation between NRAGE nuclear protein and β-catenin nuclear protein were analyzed using the Mantel–Haenszel chi-squared test. Survival data were evaluated using univariate log-rank test and multivariate Cox regression analyses. These data were analyzed using SPSS 25 statistical software . A P-value <0.05 was considered statistically significant.vs EN, 0Gy: p5d <0.0001, p6d <0.0001, p7d <0.0001; 2 Gy: p4d = 0.0277, p5d <0.0001, p6d <0.0001, p7d <0.0001; 5 Gy: p4d <0.0001, p5d <0.0001, p6d <0.0001, p7d <0.0001; p = 0.0429) and increased radiobiological parameter, SF2 (E vs EN = 0.518 vs 0.636), D0 (E vs EN = 1.201 vs 2.020), and Dq (E vs EN = 1.492 vs 1.530), under a series of doses of 0, 2, 4, 6, 8, and 10 Gy . After RT, E/N cells had lower apoptosis than E cells  (p = 0.01), the most radioresistant cell stage, and a lower ratio in the most radiosensitive cell stage G2/M . After treatment with 5 Gy IR, cell cycle distributions were rearranged to a greater extent with more arrested cells in the S phase  and G0/G1 phase  and downregulated cells in the G2/M phase . These revealed that NRAGE overexpression could reduce cell apoptosis and change cell cycle division of ESCC, affecting cellular radioresistance.To further define the mechanism of NRAGE in ESCC radioresistance, cell migration, invasion, cell cycle progression, and apoptosis in E/N cells were analyzed in addition to the detection of proliferation. Wound healing assays were performed in E and E/N cells with or without 5 Gy X-ray radiation and then imaged at 12 and 24 h. Results showed that ESCC cells with upregulated NRAGE had significantly faster migration ratio than E cells, especially after IR (p= 0.037) level, followed by increased trend of p-Gsk-3β levels (p = 0.917), led to the increased expression of cyclin D1 (p = 0.023), a targeting gene of β-catenin in E/N cells, while downregulation of p-β-catenin (p = 0.037) and Gsk-3β (p = 0.049) was detected , 2 h , 6 h  , β-catenin  and p-Gsk-3β  decreased, while p-β-catenin  and Gsk-3β  slightly increased after FH535 treatment  was used as the 3D bioprinted material. Hydrogel seeded with E and E/N cells were extruded at variable pressure (0.31 MPa), needle type , and needle diameter (340 µm) to study cell characteristics directly after printing. Extruded gelatin–alginate blend was stained with live/dead dye and imaged   and in ESCC samples compared with paired Paracancerous tissue from the GSE20347 dataset  (p = 0.0001) (p = 0.0021) (p = 0.081) (p = 0.0037)  compared with adjacent normal tissue samples (286 cases) (ov/geo/) Table S2p = 0.0023, p = 0.006). However, regardless of NRAGE total protein or NRAGE nuclear protein, there was no association with age , gender , clinical stage , tumor size , LNM , synchronous chemotherapy , and events  , sex , clinical stage , tumor size , LNM , synchronous chemotherapy , and events  was found. A significant correlation could not be found between β-catenin total protein expression and clinicopathological features (p = 0.143), but a strong association between β-catenin nuclear protein expression and curative efficacy was observed (p = 0.006). Kaplan–Meier survival curves exhibited no association between OS in definitive RT and NRAGE or β-catenin total protein expression , but a strong positive NRAGE nuclear protein expression was significantly shorter than those with positive and weak positive NRAGE expression (p <0.0001). Additionally, there was a correlated trend between β-catenin nuclear protein expression and OS (p = 0.081). Moreover, we analyzed the association between NRAGE and β-catenin nuclear protein expressions and found their linear correlation trend  (p = 0.037), curative efficacy (p = 0.000), and strong positive NRAGE nuclear protein expression (p = 0.000) were associated with a significantly increased risk of death in patients with ESCC receiving radical RT . Synchronously, clinical stage  and synchronous chemotherapy  were included as factors  Table 1.EC is one of the most common primary malignancies with high mortality, and mainly in ESCC in China. RT is one of the primary therapeutic modalities in patients with ESCC. The existence of radioresistance is a major limit to achieve long-term survival in ESCC, which has been linked to an increased likelihood of recurrence and distant metastasis –32.NRAGE has complicated and contradictory functions. It encodes an 86-KDa protein and is a member of the Type II MAGE family, comprising 778 amino acids (aa), of which the MAGE homology domain is common for MAGE family members and the interspersed repeat domain (IRD) is relatively unique to NRAGE for no homology to any public specific protein. These features imply that there are both uniform and specific characteristics of NRAGE compared with other members of the MAGE family –36. GrowInnovatively, our team provided a new insight for NRAGE into the ability of pro-radioresistance that NRAGE was unexpectedly overexpressed in radioresistant ESCC cells based on gene microarray analysis and experiment verification. It was shown that NRAGE had a growing trend following enhanced IR dose and time . Anotherin vivo but also shorten the research cycle compared with the mouse model  for the publication of any potentially identifiable images or data included in this article.HZ and GW is attributed for the study design, data analysis, and manuscript writing. ZX established cell lines, tissue collection, and evaluation of radiotherapy efficacy. ZX, YY, and ZT helped interpreting the data. CG, XH, WS, and LH prepared all the figures. JL, HZ, and GW edited all the tables. XX is attributed for the experiment conduction. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work is supported by a grant from the Natural Science Foundation of Hebei Province (No. H2019206182).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Integration of these advantageous components enables the imaging of the activity of neuronal ensembles in awake mice minimally invasively with excellent signal-to-background and subsecond temporal resolution. This study thus establishes a paradigm for studying brain function in health and disease.Although fluorescent indicators have been broadly utilized for monitoring bioactivities, fluorescence imaging, when applied to mammals, is limited to superficial targets or requires invasive surgical procedures. Thus, there is emerging interest in developing bioluminescent indicators for noninvasive mammalian imaging. Bioluminescence imaging (BLI) of neuronal activity is highly desired but hindered by insufficient photons needed to digitalize fast brain activities. In this work, we develop a luciferase prosubstrate deliverable at an increased dose and activated in vivo by nonspecific esterase. We further engineer a bright, bioluminescent indicator with robust responsiveness to calcium ions (Ca Bioluminescence imaging of neuronal activity is desired. Here the authors report a luciferase prosubstrate activatable in vivo by nonspecific esterase and engineer a bioluminescent indicator with responsiveness to calcium ions: integration of these components enabled imaging of neuronal activity in mice. Despite the progress, fluorescence neuronal imaging is practically invasive and only reaches a shallow depth. Cranial windows or thinned skulls are often used to access the cortex. Due to tissue absorption and scattering, the imaging depth is ~200 μm for widefield one-photon and a couple of millimeters with multiphoton excitation5. To reach deeper brain regions, more invasive procedures, such as implanting optical fibers or gradient-index (GRIN) lenses, are needed3.Fluorescence imaging is the standard method for following brain activity in small behaving animals. Genetically encoded fluorescent indicators, including GCaMPs and other Ca7. Because bioluminescence needs no excitation, photons emitting from the embedded light sources can travel through several centimeters of mammalian tissue8. In addition, compared to fluorescence, BLI has a low background, no photobleaching and phototoxicity, and minimized disturbances of light-sensitive biological components 12. Moreover, BLI has excellent compatibility with popular optogenetic and optochemical tools13. Macroscopic BLI only requires a dark box with a sensitive camera. Similar setups are already in many laboratories and core facilities; otherwise, they are commercially available or can be constructed at affordable costs.Bioluminescence, which refers to photon emission from luciferase-catalyzed exothermic oxidation of the corresponding luciferin, is a promising imaging modality for noninvasive in vivo recording7. Insect luciferases are generally slow in catalysis and consume adenosine triphosphate (ATP) for luciferin activation and photon production. In contrast, the oxidation of the coelenterazine  for redder emission via bioluminescence resonance energy transfer (BRET)20.Commonly used luciferase-luciferin pairs originate from either insects or marine organisms2+ is a ubiquitous second messenger, and intracellular Ca2+ has been used as a proxy for neuronal activity3. Previous studies have used Ca2+-sensitive photoproteins, such as aequorin, obelin, and their mutants, for Ca2+ detection, but they have drawbacks, including weak light emission and irreversible responses due to slow luciferin recharging23. Other studies introduced Ca2+-sensory elements into luciferases, including NanoLuc and NanoLuc-FP hybrid reporters, resulting in reversible Ca2+ indicators with much-increased light production27.Bioluminescent indicators that change signals in response to neuronal activity are needed for functional imaging. Ca29. Another limiting factor is the low amount of marine luciferase substrates that can be systematically delivered to the brain31. Moreover, most current bioluminescent Ca2+ indicators  and M13 moieties between residues 133 and 134 of NanoLuc linked to two copies of CyOFP1 (an orange-emitting FP), are the only NanoLuc-based indicators with appreciable emission above 600 nm29. The Orange CaMBI 110 (OCaMBI110) variant has been successfully used to image in vivo Ca2+ dynamics in the mouse liver but not yet in the brain29.Despite the progress, functional BLI (fBLI) of neuronal activity, which requires fast digitalization, is still hindered by insufficient photons reaching detectors. First, the intrinsic photon production rates of NanoLuc and NanoLuc-based indicators remain several orders of magnitude lower than those achievable in typical fluorescence imaging setups2+ responsiveness. We characterize this integrated system for imaging Ca2+ dynamics in cultured cell lines, primary mouse neurons, and acute brain slices. Moreover, because of the drastically increased photon flux, the integrated system enables the minimally invasive imaging of deep-brain Ca2+ dynamics in awake mice in response to behavioral and disease triggers.To fill the fBLI technical gap, we develop a luciferase prosubstrate for enhanced luciferin delivery and a bioluminescent indicator with redder emission and remarkable brightness and Ca15. DTZ could be synthesized from inexpensive commercial reagents in two steps with excellent yields  and ABCG2, were shown to pump out CTZ34. The BBB efflux issue is further compounded by additional unfavorable factors, such as the rapid clearance and low solubility of these substrates35. The literature reported that adding a succinate group to paclitaxel (Taxol) reduced the interaction with P-gp and increased brain distribution vastly37, because P-gp unfavorably interacts with negatively charged molecules and the succinate addition installs a carboxylate functional group with a pKa of ~4. Thus, we chose to use the carboxylate functional group to modify DTZ with the hope of enhancing its hydrophilicity, reducing BBB efflux, and increasing possible dosage via aqueous intravenous injection buffers.Our group previously reported teLuc, a bright and red-shifted NanoLuc mutant, and its paired DTZ substrate . In addition, for comparison purposes, we synthesized C3-DMA-DTZ  mScarlet-I, resulting in a LumiScarlet reporter with ~ 51% of the total emission above 600 nm16. Following the success, we created a similar fusion between teLuc and mScarlet-I and optimized the linker for increased BRET efficiency  with ~60% of its total emission above 600 nm  are less absorbed and scattered by mammalian tissue, thus penetrating tissue deeper nm Fig. . AlthougWe next compared DTZ, ETZ, and C3-DMA-DTZ for bioluminescence in cultured mammalian cells. We transiently expressed BREP in human embryonic kidney (HEK) 293 T cells and imaged the cells in 96-well plates upon adding 25 μM of each compound  with the BREP gene driven by the human synapsin I (hSyn) promoter and transduced hippocampal neurons in live mice via stereotactic injection. We tested the BREP-expressing mice against a panel of CTZ analogs  of ETZ and DTZ. Although mice in the ETZ group seemed slightly brighter than those in the DTZ group, there was inadequate statistical power to resolve the difference  Ca2+-dependent bioluminescence increase  with a 6.5-fold (BL/BL0) response  of BRIC to Ca2+ was determined to be 133 nM  into the hippocampus of BALB/cJ mice Fig.  and comp2+ dynamics in awake mice  in the brain in live mice51. Particularly, a substrate manufactured by Promega Corporation, namely ViviRen, was shown to improve the light output of Renilla luciferase in live mice bearing brain tumors compared to the equimolar amount of CTZ30. However, these previously explored C-3 caging groups were typically hydrophobic, unfavorably reducing substrate solubility in aqueous solutions. Also, a prosubstrate strategy was recently used to deliver the amide derivatives of firefly luciferins into the mouse brain for fatty acid amide hydrolase (FAAH)-dependent activation53.Of note, several other studies developed C-3 caged CTZ or FRZ analogs, some of which also slowed bioluminescence decays in living cells55.BREP is the most red-shifted marine luciferase-FP fusion protein that retains high brightness. The construct uses mScarlet-I as the BRET acceptor. Because existing far-red and near-infrared FPs that are more red-shifted than mScarlet-I have much-reduced fluorescence quantum yields, it is not yet possible to use the FP fusion strategy to further red-shift bioluminescence without remarkably harming brightness. Alternatively, one may covalently conjugate far-red or near-infrared synthetic dyes to luciferase variants via self-labeling protein tags 2+ indicator. We compared it with the current benchmark, OCaMBI110. BRIC showed larger responses to Ca2+ as purified proteins and in HeLa cells and primary neurons. In addition, BRIC was less prone to aggregation. When applied in mice, the BRIC signals in the presence of ETZ were much brighter and lasted much longer than OCaMBI110 with either FRZ or FFz. The integration of BRIC with ETZ allowed us to image Ca2+ dynamics in awake mice minimally invasively with excellent signal-to-background and subsecond temporal resolution. However, despite the enhancement, BRIC is still suboptimal since its response to physiological Ca2+ changes  is limited to ~2-fold. We plan to further optimize the practical dynamic range of this indicator in future.BRIC is a high-performance and red-shifted bioluminescent Ca2+ signals after the action potential is likely within this time range, the kinetics of Ca2+ concentration changes is much faster than the achieved temporal resolution56. Thus, interested users should be aware that the recorded signals are undersampled over time. Considering these reasons and the fact that fluorescent Ca2+ indicators, such as GCaMPs, have been extensively optimized56, fluorescence remains the choice of imaging modality for fiber photometry or single-cell-resolution recording through cranial windows. On the other hand, the method presented here offers technical simplicity, reduced invasiveness, and deep-brain imaging capability. The approach complements, but cannot replace, fluorescence or electrical recording.Our current approach macroscopically records concerted changes in a neuronal population that expresses the BRIC indicator. The spatial resolution is limited by scattering when photons travel through brain tissue, skull, and skin. Also, photons reaching the detector are still scarce; when using the experiment setups presented here, the temporal resolution is in the second and subsecond ranges. Although the overall duration of the rising and extinction of intracellular Ca58. We are currently exploring these AAV vectors for peripheral gene delivery and transduction of specific neuronal populations in transgenic Cre mice with the C57BL/6 genetic background. We expect the effort to lead to an utterly noninvasive strategy for recording the activity of neuronal ensembles in small mammals.This study tested ETZ in both BALB/cJ and C57BL/6 J strains of mice and used intracranial injection to deliver viral vectors. Recently, AAV vectors with engineered capsids have been reported to cross the BBB of C57BL/6 J mice and marmoset2+ changes. Future developments may allow two-channel BLI of the mouse brain with high sensitivity, enabling ratiometric correction for baseline decays, blood flow changes, and animal motions.Also, we had to apply baseline corrections for time-lapse intensity quantitation. A monoexponential decay model was adequate for baseline corrections in those experiments spanning short periods, although a more complex decay was evident during the more extended 60-min period shown in Fig. 31. To gain enough signals, a cranial window was used for bioluminescence collection and continuous substrate delivery to the cortex. Furthermore, a GaAsP image intensifier was placed in front of the EMCCD to boost signals. Another recent preprint described a NanoLuc-derived bioluminescent glutamate indicator that has been tested in cultured cells59. We expect our results and strategies presented here to assist in the further development of these bioluminescent indicators for deep-brain in vivo imaging with minimally invasive procedures. Furthermore, as much-enhanced BLI tools, ETZ, BREP, and BRIC are expected to find diverse applications and generate broad impacts beyond neurobiology.In addition, a recent study described the use of FRZ and a NanoLuc-derived bioluminescent voltage indicator for imaging cortical activity in miceThe study complies with all relevant ethical regulations, and all animal experiments were conducted according to the approval (Protocol #4196) and guidelines of the University of Virginia Institutional Animal Care and Use Committee. BALB/cJ mice (#000651) and C57BL/6 J mice (#000664) were purchased from the Jackson Laboratory and bred under standard conditions. Mice were hosted in a temperature-controlled room (~23 °C) with a 12 h/12 h dark-light cycle and ~50% humidity. Animals were randomly allocated to experimental groups with a balance of females and males at the age of ~8 weeks.124094) and pAAV.hSyn.iGluSnFr.WPRE.SV40 (Addgene #98929) were gifts from M.Z. Lin and L.L. Looger, respectively. pAdDeltaF6 (Addgene #112867), pAAV2/9n (Addgene #112865) were gifts from J.M. Wilson. Synthetic DNA oligonucleotides (Supplementary Table 3) or 3.30 (methanol-d4) ppm for 1H-NMR, and 77.2 (CDCl3) or 49.0 (methanol-d4) ppm for 13C-NMR. Splitting patterns of NMR active peaks are reported as s (singlet), d (doublet), t (triplet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). The BMG Labtech CLARIOstar Plus Reader Software (Version 5.70 R2) was used to control the CLARIOstar Plus microplate reader, and results were automatically exported to the BMG Labtech MARS Data Analysis Software (Version 3. 42 R5) for analysis. Agilent MassHunter Workstation Data Acquisition (Version B.09) was used to acquire high-resolution mass spectra (HR-MS) on an Agilent 6545 Q-TOF LC/MS system via direct infusion. Agilent MassHunter Quantitative Analysis Navigator (Version B.08) was used to analyze HR-MS data. The UVP VisionWorksLS software (Version 8.6) and μManager (Version 2) were used to control a UVP BioSpectrum dark box and cameras for macroscopic BLI, while Leica LAS X (Version 3.5.7) or MicroManager (Version 2.0) was used to acquire microscopic images. The ChemAxon LogD Predictor  was used to predict the logD values of the specified compounds at pH 7.Unless otherwise stated, all chemicals were purchased from MilliporeSigma, Fisher Scientific, or VWR and used without further purification. Kainic acid monohydrate (KA) was purchased from Cayman Chemical. pcDNA3.1-Orange_CaMBI_110  was added into 30 mL anhydrous THF under argon (Ar) protection. Subsequently, BnMgCl  was dropwise added into the reaction mixture in a dry ice-acetone bath. The mixture was stirred for 1 h in the dry ice-acetone bath, and then at room temperature for another 2 h. The reaction was next quenched by addition of H2O. The mixture was filtrated and extracted three times with EtOAc. The organic layer was combined, dried with anhydrous Na2SO4, concentrated in vacuo. Purification was performed with silica gel chromatography  to give compound 1 .Compound 2SO4. After filtration and rotovap concentration, the residue was further purified with silica gel chromatography  to afford compound 2 .NHS , DMAP , TEA  and propargyl alcohol  were separately added into a solution of succinic anhydride  dissolved in 50 mL anhydrous toluene. The mixture was heated to reflux overnight under Ar. After cooling, the mixture was washed with brine and extracted three times with EtOAc. The organic layer was combined and dried with anhydrous Na3 was synthesized by modifying a previous method15. 2-Amino-3,5-dibromopyrazine , phenylboronic acid  and bis(benzonitrile)dichloro palladium  were dissolved in EtOH. Next, 1 N Na2CO3  was added into the reaction mixture, which was further heated to reflux overnight under Ar. After cooling down to room temperature, the mixture was filtered, and EtOH was removed with rotovap. The residue was acidified to pH 4~5 with HCl (1 N) and washed three times with EtOAc. The aqueous layer was next alkalized to pH ~11 with NaOH (1 N) and extracted three times with EtOAc. The organic layer was combined, washed with brine and H2O three times, dried with anhydrous Na2SO4, filtered, and concentrated in vacuo to give compound 3 .Compound 15. 6 N HCl  was added to 5 mL of 1,4-dioxane in a 60-mL Ace pressure tube (Sigma-Aldrich #Z568767) containing compound 3  and 1,1-diethoxy-3-phenylacetone . The tube was sealed, and the mixture was maintained at 120 °C with stirring overnight. Next, the mixture was cooled down to room temperature before the solvent was removed under reduced pressure. The crude was re-dissolved in 15 mL MeOH, and next, purified with the Waters preparative RPLC-MS . Product fractions were combined and lyophilized to give DTZ .DTZ was synthesized by modifying a previous method2  and DCC  were placed in an oven-dried two-neck round-bottom flask purged with Ar three times. Anhydrous DCM (10 mL) mixed with TEA  and two drops of anhydrous DMF was injected into the reaction system with stirring at room temperature over 15 min. Subsequently, DTZ  was quickly added into the reaction system through one neck of the flask. The system was flushed with Ar and the reaction mixture was stirred for additional 20~30 min. The progress of the reaction was monitored with TLC (hexane/EtOAc = 3:1). After completion of the reaction, the mixture was cooled down to −20 °C to precipitate the DCU by-product. After filtration and concentration in vacuo, the residue was further purified with silica gel chromatography  to give compound 4 . 1H NMR  δ 8.89–8.84 , 8.24 , 8.16 , 7.60–7.55 , 7.54–7.46 , 7.42–7.37 , 7.36–7.29 , 7.26–7.22 , 4.75 , 4.24 , 2.79–2.74 , 2.73–2.64 , 2.46 . 13C NMR  δ 171.7, 169.0, 147.8, 138.5, 138.1, 136.8, 136.1, 135.3, 133.3, 130.4, 130.2, 129.8, 129.2, 128.8, 128.7, 128.5, 128.4, 128.3, 126.4, 126.3, 109.7, 77.2, 75.5, 75.3, 52.6, 34.3, 28.8, 28.1. ESI-MS (m/z): [M + H]+ calcd. for C32H25N3O4, 516.19; found, 516.18.Compound 4 , CuI  and THPTA  were placed in a two-neck round-bottom flask purged with Ar three times. Azidoacetic acid  was dissolved in THF (10 mL) and injected into the reaction system. Next, ascorbic acid  dissolved in 2 mL ddH2O was dropwise injected into the reaction mixture and stirred overnight at room temperature. After completion of the transformation, the mixture was filtered and the product was purified with the Waters preparative RPLC-MS . Product fractions were combined and lyophilized to give ETZ . 1H NMR  δ 8.69–8.64 , 8.51 , 8.16 , 7.92 , 7.57–7.52 , 7.45 , 7.39–7.34 , 7.33–7.23 , 7.22–7.17 , 5.28 , 5.15 , 4.14 , 2.87–2.84 , 2.81–2.77 .13C NMR  δ 178.2, 172.6, 169.7, 147.6, 142.3, 138.5, 138.1, 136.5, 135.9, 135.5, 132.9, 130.0, 129.4, 128.9, 128.7, 128.4, 128.1, 127.9, 126.1, 126.0, 125.8, 124.9, 110.1, 57.4, 50.4, 32.8, 28.3, 27.9. ESI-HRMS (m/z): [M + H]+ calcd. for C34H28N6O6, 617.2149; found, 617.2149.Compound in vacuo. The residue was dissolved in MeOH: ddH2O  and purified with the Waters preparative RPLC-MS . Product fractions were combined and lyophilized to give crude compound 5 .NHS , DMAP , TEA  and 2-dimethylaminoethanol  were consequently added into a solution of succinic anhydride  dissolved in anhydrous toluene (50 mL). The mixture was heated to reflux overnight under Ar. After cooling to room temperature, the solvent was removed 1H NMR  δ 8.68–8.64 , 8.52 , 8.18–8.14 , 7.58–7.51 , 7.48–7.44 , 7.40–7.36 , 7.32–7.26 , 7.23–7.18 , 4.28 , 4.16 , 2.88–2.84 , 2.83–2.78 , 2.66–2.62 , 2.27 .13C NMR  δ 173.2, 172.5, 130.0, 129.8, 129.4, 129.3, 128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 126.2, 126.1, 126.0, 125.6, 125.5, 125.2, 58.2, 56.9, 43.9, 42.7, 32.1, 29.3, 28.2. ESI-HRMS (m/z): [M + H]+ calcd. for C33H32N4O4, 549.2502; found, 549.2496.Compound 5  and DCC  were placed in an oven-dried two-neck round-bottom flask purged with Ar three times. Anhydrous DCM (10 mL), DMF (1 mL), and TEA  were immediately added into the reaction mixture with stirring at room temperature over 30 min. Subsequently, DTZ  was added to the reaction mixture. The resulting mixture was stirred for additional 80 min, then filtered, concentrated, and purified with silica gel chromatography  to give C3-DMA-DTZ .15 and pcDNA3-teLuc-myc16 were used as separate PCR templates. The mScarlet-I fragment was amplified from pcDNA3-LumiScarlet with the forward primer pBAD_FW_EP and one of the three reverse primers—pBAD_RV_BREP1_NNK1, pBAD_RV_BREP1_NNK2, and pBAD_RV_BREP1_3NDT . Cells were allowed to grow on 2 × YT agar plates supplemented with 100 μg/mL ampicillin and 0.02% (w/v) L-arabinose at 37 °C overnight. Single colonies were selected for growth in 2×YT liquid culture. Plasmids were recovered and Sanger sequencing confirmed the successful creation of pBAD-BRIC0.1. Next, Taq DNA polymerase (New England Biolabs) in the presence of MnCl2 (0.05 mM) or GeneMorph II Random Mutagenesis Kit (Agilent Technologies) was used for error-prone PCR (EP-PCR)-based random mutagenesis. Oligos pBAD_FW_EP and pBAD_RV_EP were used for these reactions. PCR products were inserted into pBAD/HisB between Xho I and Hind III via Gibson assembly. The DNA libraries were used to transform E. coli DH10B cells, which were cultured on 2 × YT agar plates supplemented with 100 μg/mL ampicillin and 0.02% (w/v) L-arabinose at 37 °C overnight. Red colonies were selected and used to inoculate cultures in 96-well deep-well bacterial culture plates. Each well was filled with 1 mL of 2 × YT broth supplemented with 100 μg/mL ampicillin and 0.2% (w/v) L-arabinose. Cells were grown at 30 °C, 250 rpm for 48 h, pelleted by centrifugation, and lysed with 300 μL of B-PER at 4 °C for 30 min. After centrifugation, cell lysates were prepared. 5 μL of the supernatant was diluted with 185 μL of a Ca2+-free buffer  and a Ca2+-containing buffer  expected to provide 39 µM free Ca2+. Next, DTZ was dissolved in the in vitro assay buffer described above to the concentration of 500 μM, and 10 μL of the DTZ solution was injected into each well via a reagent injector in a BMG Labtech CLARIOstar Plus microplate reader. The final concentration of DTZ was thus 25 μM. The bioluminescence spectrum and intensity of each well were measured from 400 to 700 nm with 5-nm intervals. Mutants with high brightness and Ca2+ responsiveness were selected.The CaM-M13 fragment was PCR-amplified from pcDNA3.1-Orange_CaMBI_110 with oligos pBad_FW2_CaM and pBad_RV2_M13. Next, pBAD-BREP was amplified with either pBad_FW1_mScarlet and pBad_RV1_teLuc(133), or pBad_FW3_teLuc(134) and pBad_RV3_teLuc(168) to generate two fragments. The three fragments, along with pBAD/HisB predigested with Xho I and Hind III, were assembled in a four-part Gibson assembly reaction. The product was used to transform 16. The purified protein was diluted with the in vitro assay buffer to a final concentration of 10 nM. 100 μL of the diluted protein was added into each well of a 96-well plate. 100 μL of 25 μM DTZ pre-dissolved in the in vitro assay buffer was injected into the well via a reagent injector in a BMG Labtech CLARIOstar Plus microplate reader. The mixture was shaken for 2 s before the bioluminescence spectrum was recorded using the equipped red-sensitive PMT. The instrument was set to scan from 400 to 700 nm with 5-nm intervals. Three technical repeats were performed to derive the average spectrum.BREP was expressed and purified following a previous procedureE. coli DH10B cells, which were next cultured on 2 × YT agar plates supplemented with ampicillin (100 μg/ml). A single colony was selected and cultured in 5 mL 2 × YT broth supplemented with 100 μg/mL ampicillin. The culture was shaken at 250 rpm and 37 °C overnight. The culture was next diluted with 500 mL of 2 × YT medium containing 100 μg/mL ampicillin. After shaking incubation at 37 °C for 2 h, protein expression was induced by adding L-arabinose , and the culture was maintained at 250 rpm and 16 °C for 96 h. Cells were pelleted by centrifugation and resuspended in 10 mL of 1× phosphate-buffered saline  supplemented with a cOmplet Mini EDTA-free Protease Inhibitor tablet (Roche). Cells were lysed by sonication, and the lysate was clarified by centrifugation at 15,000 × g for 30 min at 4 °C. The His6-tagged protein was enriched with and then eluted from Ni-NTA agarose beads (Genesee Scientific). Finally, the protein was subjected to a size-exclusion HiLoad 16/600 Superdex 200 pg column (Cytiva) and eluted with an aqueous buffer containing 150 mM NaCl and 30 mM Tris HCl, pH 7.4.pBAD-BRIC was used to transform 6-tagged protein in cell lysates was first enriched with Ni-NTA agarose beads, and next, the eluate was applied to a Strep-Tactin Superflow high-capacity column (IBA Lifesciences). Finally, the eluate from the Strep-Tactin column was subjected to size-exclusion chromatography. A similar pBAD-BRIC-Step plasmid was constructed in parallel. The spectral properties of the BRIC protein prepared from pBAD-BRIC-Step with dual His6 and Strep tags were identical to the protein prepared from pBAD-BRIC only with the N-terminal His6 tag.A pBAD-OCaMB110 plasmid was created by amplifying the indicator gene from pcDNA3.1-Orange_CaMBI_110, but it turned to be challenging to prepare high-purity OCaMB110 protein due to proteolysis. To address the issue, an additional Strep-tag II sequence was appended to the C-terminus of the reading frame, resulting in a pBAD-OCaMB110-Step plasmid. Protein expression was performed with the identical procedure described above. To purify the protein, the N-terminal His2+-containing and Ca2+-free buffers mentioned above were used to record the emission spectra for Ca2+-free and Ca2+-saturated states. Final protein and substrate concentrations were 50 nM and 25 μM, respectively. The Ca2+ affinity was determined as previously described using a series of buffers made from mixing the Ca2+-containing and Ca2+-free buffers61. The bioluminescence intensity at 595 nm recorded on a BMG Labtech CLARIOstar Plus microplate reader was plotted against the expected free Ca2+ concentration in each buffer. Three technical repeats were performed, and the data was fit with a one-site binding model in GraphPad Prism. To determine pH sensitivity, the Ca2+-containing and Ca2+-free buffers were adjusted with HCl (12 M and 1 M) or KOH (4 M) to gain buffers with pH values ranging from 5.5 to 8.0. Bioluminescence intensity ratios at 595 nm in the presence and absence of Ca2+ were plotted against the pH values.Protein concentrations were determined with the Pierce 660 nm Protein assay using bovine serum albumin (BSA) standards. The Ca16. Cells were collected 20 h post-transfection, and resuspended in and diluted with 1×PBS (pH 7.2). ~7,000 cells in 100 μL of total volume were placed in individual wells in a 96-well plate. The compounds  were dissolved in 1×PBS (pH 7.2) to gain 50 μM concentrations. Next, 100 μL of the substrates were added to BREP-expressing HEK 293 T cells in the 96-well plate. BLI was performed immediately with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. The camera was first set to the “Photon Counting” mode using OptAcquire pre-settings, and the gain was next adjusted down to 500. Other parameters were: camera binning 1 × 1; camera sensor temperature −70 °C; 100 ms exposure time with acquisitions every 10 s. The lens was controlled using the UVP VisionWorksLS software with aperture set to f12.8, zoom set to 32%, and focus set to 0%. The plate was placed 27 cm away from the front of the lens with no emission filter. Images were acquired with the μManager software and processed in the Fiji version of ImageJ 2.1. Image stacks were first subtracted for background by setting the rolling ball radius to 100 pixels. Next, the region of interest (ROI) was selected by circling cell locations, and the intensity value integrated over the ROI was extracted for further analysis. Data were plotted and statistical analysis was performed in GraphPad Prism. After the software-based background subtraction, the images were left with residual background, so the ROI was moved away from the cell locations to evaluate residual background, which was further used to subtract signals for calculating the integrated bioluminescence intensity (area under the curve).pBad-BREP was amplified with oligos pcDNA3_FW_HindIII and pcDNA3_RV_XhoI via PCRs. The fragment was inserted into pcDNA3 to afford pcDNA3-BREP. pcDNA3-BREP was next used to transfect HEK 293 T cells  using a described procedure62. was followed for viral packing and purification. Viral titers were determined with quantitative PCR (qPCR) based on SYBR green by following a protocol from Addgene. Typical AAV titers were 5 × 1014 − 1 × 1015 GC/mL. AAVs were aliquoted and stored at −80 °C for long-term stability.Indicator genes were amplified from their corresponding pcDNA3/3.1 plasmids and inserted into a pAAV-hSyn vector to generate pAAV-hSyn-BRIC, pAAV-hSyn-BREP, and pAAV-hSyn-OCaMBI110. Next, individual transfer plasmids, along with pAdDeltaF6 and pAAV2/9n, were used to transfect HEK 293 T cells to pack viruses. A protocol by Rego et al.63 of 8-week-old, anesthetized BALB/cJ mice via intracranial stereotactic injection at a flow rate of 200 nL min−1. When the infusion was complete, the needle was kept in the brain for 5 min before being withdrawn. The wound was then sealed with surgical adhesive.Cells were cultured, transfected, and collected as described in the above section. 3 μL of cells  were delivered into each side of the hippocampus 3; a 5 mL injection buffer for C3-DMA-DTZ was made by dissolving 1.25 g HP-β-CD and 1 mL PEG-400 in ~3 mL of normal saline pre-supplemented with 100 mM citric acid (pH 5.5). NaHCO3 and citric acid have been used broadly for intravenous infusion and were supplemented to formulate ETZ and C3-DMA-DTZ as salts in situ. This buffer system was modified from the previously used recipe19 with remarkably reduced viscosity, allowing more consistent intravenous injection.Meanwhile, the compounds DTZ, ETZ, and C3-DMA-DTZ were dissolved in aqueous buffers supplemented with 25% (w/v) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and 20% (v/v) PEG-400. More specifically, a 5 mL injection buffer for DTZ was made by dissolving 1.25 g HP-β-CD and 1 mL PEG-400 in ~3 mL normal saline; a 5 mL injection buffer for ETZ was made by dissolving 1.25 g HP-β-CD and 1 mL PEG-400 in ~3 mL of normal saline pre-supplemented with 5% (w/v) NaHCO100 μL of DTZ (2.5 mM), ETZ (6.8 mM), or C3-DMA-DTZ (4.5 mM) in their corresponding in vivo injection buffers was delivered to mice via tail vein right after intracranial cell injection. Bioluminescence images were acquired with a BLI system consisting of a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3) and a QSI 628 Cooled CCD camera. The settings were: camera binning 4 × 4; high gain; camera sensor temperature −15 °C; 10 s exposure time and each acquisition every 60 s. The lens was controlled using the UVP VisionWorksLS software with aperture set to 100% open, zoom set to 0%, and focus set to 0%. Anesthetized mice were placed 21 cm away from the front of the lens with no emission filter used. To minimize biological variables, each mouse was sequentially tested with three substrates, and the next substrate was administered after the signal of the previous substrate faded out. A total of three mice were used, and the substrate injection order was rotated for each mouse to control the bias.15 GC/mL) was delivered to each side of the hippocampus of 8-week-old BALB/cJ mice via intracranial stereotactic injection at a flow rate of 100 nL min−1 using the coordinate described above. Three weeks later, mice were used for brightness comparison. ETZ (6.8 mM), ETZ (2.5 mM), DTZ (2.5 mM), C3-DMA-DTZ (4.5 mM), FFz (6.0 mM), and FRZ (3.0 mM) were dissolved in their corresponding in vivo injection buffers, and 100 μL of each substrate was delivered to mice via tail vein. BLI was subsequently performed with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. The camera was first set to the “Photon Counting” mode using OptAcquire pre-settings, and the gain was next adjusted down to 500. Other parameters were: camera binning 2 × 2; camera temperature −70 °C; 500 ms exposure time with acquisitions every 10 s. The lens was controlled using the UVP VisionWorksLS software with aperture set to 100% open, zoom set to 0%, and focus set to 0%. Anesthetized mice were placed 27 cm away from the front of the lens with no emission filter used.To further evaluate the compounds with endogenous neurons in mice, 500 nL of BREP AAV  was selected based on bioluminescence from the mouse brain, and the intensity value integrated over the ROI was extracted for further analysis. Data were plotted and statistical analysis was performed in GraphPad Prism. After the software-based background subtraction, the images were left with residual background, so the ROI was moved away from the mouse brain region to evaluate residual background, which was further used to subtract signals for calculating the integrated bioluminescence intensity (area under the curve).16. Cells were allowed to grow at 37 °C in a 5% CO2 incubator for 18 h. Cells were rinsed twice with 1 × PBS and placed in 1 × PBS 15 min before imaging. Images were acquired on an inverted Leica DMi8 microscope equipped with a Photometrics Prime 95B Scientific CMOS camera. 40 µM DTZ or 40 µM FRZ was supplemented for bioluminescence. Imaging settings were: 40× oil immersion objective lens (NA 1.2), no filter cube, 2 × 2 camera binning, 1 s exposure with 0 s interval, camera sensor temperature −20 °C; 12-bit and high sensitivity mode. Histamine was dissolved in 1 × PBS and added during time-lapse imaging to a final concentration of 100 μM. Image stacks were processed as described in the previous section, except that ROIs were selected for individual cells and the mean of intensity values over the ROI was extracted for further analysis. Moreover, the baselines caused by substrate decay were fitted to a monoexponential model: Y = (Y0-Plateau)*exp(-K*X) + Plateau. Data were plotted and statistical analysis was performed in GraphPad Prism.pBad-BRIC was used for PCRs with oligos pcDNA3_FW_HindIII and pcDNA3_RV_XhoI. The fragment was inserted into pcDNA3 to afford pcDNA3-BRIC. HeLa cells  were transfected with 3 μg of the plasmid pcDNA3-BRIC or pcDNA3.1-Orange_CaMBI_110 using a described procedure2. Half of the medium was changed to fresh NbActiv4 every 2 days. Neurons were transduced with AAVs  at the 5th day post plating. 3 µL of each virus at ~5 × 1014 GC/mL and 1 μL of 1 M HEPES (pH 7.4) were added to each 35 mm culture dish (with cells at ~ 60% confluency). Half of the medium was replaced with fresh NbActiv 24 h later and then every 2 days. Neurons were evaluated on the fourth or fifth day after AAV transduction. Because extensive fluorescent aggregations were observed in OCaMBI110-expressing neurons on day 5, neurons on day 4 after AAV transduction were used for quantitative comparison. Growth medium was replaced with 0.8 mL of the luminescence imaging buffer29  supplemented with 100 µM of DTZ or FRZ. Images were acquired on an inverted Leica DMi8 microscope equipped with a Photometrics Prime 95B Scientific CMOS camera. During time-lapse imaging, 0.2 mL of the high K+ stimulation buffer29  was added to depolarize cells. Instrumental settings and data analysis were identical to those described for the HeLa cell experiment, except that the exposure time was 2 s. To use BRIC to image ionomycin-induced Ca2+ influx, neurons on day 4 after BRIC AAV transfection were placed in 1 mL of the luminescence imaging buffer supplemented with 100 µM DTZ. During time-lapse imaging, 1 μL of 10 mM ionomycin was added to gain a final concentration of ~10 μM. The luminescence imaging buffer described above contains 2 mM Ca2+. In the experiment with 10 mM extracellular Ca2+, the luminescence imaging buffer was additionally supplemented with 8 mM CaCl2. Instrumental settings and data analysis were unchanged from the high K+ depolarization experiment.Freshly extracted embryonic day 18 primary mouse brain tissue was collected for neuron dissociation. The neurons were plated on poly-D-lysine coated 35 mm glass-bottom dishes with 2 mL NbActiv4 medium (BrainBits) at 37 °C and 5% CO15 GC/mL) was delivered to each side of the hippocampus of 8-week-old BALB/cJ mice via intracranial stereotactic injection at a flow rate of 100 nL min−1 using the coordinate described above. 19 days after viral injection, mice were utilized for brightness comparison. 100 μL ETZ (6.8 mM) in the in vivo injection buffer was intravenously delivered into the anesthetized BRIC-expressing mouse. Subsequently, BLI was performed with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. The camera was first set to the “Photon Counting” mode using OptAcquire pre-settings, and the gain was next adjusted down to 500. Other parameters were: camera binning 2 × 2; camera temperature −70 °C; 500 ms exposure time with acquisitions every 5 s. The lens was controlled using the UVP VisionWorksLS software with aperture set to 100% open, zoom set to 0%, and focus set to 0%. Anesthetized mice were placed 21 cm away from the front of the lens with no emission filter used. FRZ and FFz were dissolved in the in vivo injection buffer (the same recipe described above for dissolving DTZ). 100 μL of FRZ (3.0 mM) or FFz (6.0 mM) were delivered into each of the anesthetized OCaMBI11-expressing mice. Imaging conditions were unchanged. Data analysis  was identical to those described above for comparing the brain delivery of the substrates.500 nL AAV . Next, brain slices were recovered in ACSF at 37 °C for 30 min and next placed in 2.0 mL of the luminescence imaging buffer (described above for neuron experiments) supplemented with 100 μM of DTZ. Images were acquired on a Scientifica SliceScope Pro 1000 equipped with a Photometrics Prime 95B Scientific CMOS camera. Imaging settings were: 4× objective lens (NA 0.1), no filter cube, 2 × 2 camera binning, 2 s exposure with 0 s interval, camera sensor temperature −15 °C; 12-bit high sensitivity mode. During time-lapse imaging, 0.5 mL of the high-K+ stimulation buffer (described above for neuron experiments) was slowly added using a perfusion pump at a rate of 11.9 μL s−1. This ~ 42 s process minimized the motion of the brain slice. BREP-expressing slices were prepared and imaged using the same procedures. Image processing and data analysis were identical to those described for the HeLa cell experiment.Acute brain slices were prepared 3 weeks post viral delivery, which is described in the previous section. Freshly extracted mouse brains were pre-cooled and sliced to be 350 μm thickness in an ice-cold ACSF buffer  of 8-week-old BALB/cJ mice and each side of the hippocampus of 8-week-old C57BL/6 J mice via intracranial stereotactic injection. The coordinate for BLA was: from Bregma, AP −1.42, ML ± 3, DV −5.664. The same coordinate described above was used for the hippocampus. The viral titers for BREP and BRIC were ~5 × 1014 and ~1 × 1015 GC/mL, respectively. BLI was performed two to four weeks later. For Ca2+ dynamics in the BLA included by footshock stimuli, 100 μL ETZ (6.8 mM) in the in vivo injection buffer was administered to an awake mouse via tail vein. The animal was next mounted on a Narishige plastic mouse head holder (SRP-AM2). Subsequently, BLI was performed with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. Instrumental settings were identical to the descriptions for in vivo BRIC and OCaMBI110 brightness comparison, except that the binning was 4 × 4 and the exposure was 1 s. Mice were placed 27 cm away from the front of the lens. Each experiment consisted of 100 s for animal acclimation, followed by 13 footshock trials. Each trial begins with a 0.8-mA electric footshock (lasting for 1 s), and the intervals between footshock stimuli were 40 s. The electric shock was generated with an A-M Systems 2100 isolated pulse stimulator. For Ca2+ dynamics in the hippocampus, KA was first delivered to mice via i.p. injection at a dosage of 20 mg per kg body weight. If no seizure was observed within 2 hours, a second dose KA (5 mg per kg body weight) was delivered via i.p. injection. Finally, when an evident tremor was observed, 100 μL ETZ (6.8 mM) in the in vivo injection buffer was administered to the mouse via tail vein. Next, BLI was performed with the head-fixed animal as described above. Data analysis was identical to those described for the HeLa cell experiment, except that ROIs were selected for bioluminescence from the brain.500 nL of the virus was at a flow rate of 100 nL min100 μL ETZ (6.8 mM) in the in vivo injection buffers was delivered to 8-week-old, anesthetized BALB/cJ mice via tail vein. BLI was subsequently performed with a UVP BioSpectrum dark box, a Computar Motorized ZOOM lens (M6Z1212MP3), and an Andor iXon Life 888 EMCCD camera. Mice were placed 21 cm away from the front of the lens with no emission filter. Other imaging conditions and data analysis procedure were identical to the descriptions in the above section. The same procedure was used to examine mice with untransfected HEK 293 T cells intracranially injected into the hippocampus.0-Plateau) × exp(−K × X) + Plateau, and the intensity of each image in the stack was rescaled. This correction procedure was adequate for histamine-induced Ca2+ dynamics in HeLa and in vivo imaging of footshock- and KA-stimulated mice. The imaging data for high K+-induced Ca2+ in cultured neurons and brain slices required a more complicated correction. Because high K+ induced a relatively large, concerted intensity increase, the baseline was not properly identified when the whole stack was used for monoexponential fitting. Instead, the mean intensity values of each image in the stack were extracted and exported to Microsoft Excel. Data points during the expected peak responses were excluded, and monoexponential fitting was applied to the remaining data points. Finally, the offline-derived decay parameters were used to correct the whole image stacks in Fiji.The Fiji version of the ImageJ 2.1 was used for image processing. Image stacks were first subtracted for background by setting the rolling ball radius to 100 pixels. Next, an average image of each stack was used to generate a binary mask, which was subsequently applied to the image stack. Thus, the information for pixels within the ROI was retained, and the intensity values for other background pixels were set to 0. This conversion was necessary so that the background signals were not amplified during the subsequent baseline decay correction. Next, the whole image stacks were subjected to “photobleaching correction”. This procedure essentially fitted the image stacks with a monoexponential model: Y =  was used to analyze microscopic images. Imaging background was typically subtracted by setting the rolling ball radius to 300 pixels and more detailed procedures for imaging processing are presented in the above sections. Microsoft Excel (Version 15.21.1), GraphPad Prism (Version 8), and Affinity Designer (Version 1.10.4) were used to analyze data and prepare figures for publication. Sample size and the number of replications for experiments are presented in figure legends. No statistical methods were used to pre-determine the sample size. No data exclusions were performed. Blinding was not implemented during sensor development and in vitro and cellular characterization experiments. For in vivo footshock and KA-induced seizure experiments, the investigator who acquired and analyzed data was blinded to the group allocation during experiments. Data are shown as mean and standard deviation (s.d.) or standard error (s.e.m.), and the information is included in figure legends. Sample size and statistical methods used to calculate Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesPeer Review FileSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5