2.1.

Molecular Biology and Protein Engineering

To develop the first prototype of T-GECO1, we fused calmodulin (CaM) and the CaM-binding peptide (CBP) from ncpGCaMP6s to the mTFP1-derived FP domain of ZnGreen1.^25,26 To further improve this prototype, we used multiple rounds of directed evolution. In each round of directed evolution, we initially screened the fluorescence in the context of Escherichia coli colonies, selecting for the brightest colonies for further testing. We then cultured these variants and prepared clarified bacterial lysate using B-PER (Thermo Scientific). We measured the fluorescence spectra in the absence of ${\mathrm{Ca}}^{2+}$ (EGTA, buffered in TBS, pH 7.3) and in the presence of 10 nM and 10 mM ${\mathrm{Ca}}^{2+}$ (buffered in TBS, pH 7.3). The DNA encoding variants with improved responses and high brightness were sequenced and used as the template for the next round of library generation.

2.2.

Protein Expression and Purification

The pBAD/HisB plasmid carrying the T-GECO1 gene was used to transform chemical or electro-competent E. coli DH10B cells, which were then grown on solid media. Single colonies were used to inoculate a starter culture supplemented with ampicillin incubated at 37°C. After 4 h, L-arabinose was added to induce expression, and the culture was shaken overnight at 37°C before harvesting the bacteria by centrifugation. The bacterial pellet was resuspended in 1× TBS, lysed by sonication, and clarified by centrifugation. The cleared lysate was incubated with Ni-NTA resin, washed, and eluted. Dialysis was done into 1× TBS using centrifugal filter units. All steps were carried out at 4°C or on ice, unless specified otherwise.

2.3.

In Vitro Purified Protein Characterization

To determine the apparent affinity for ${\mathrm{Ca}}^{2+}$, buffers were prepared with varying concentrations of free-${\mathrm{Ca}}^{2+}$ ranging from zero to $39\mu \mathrm{M}$ by combining appropriate volumes of ${\mathrm{Ca}}^{2+}$-free and ${\mathrm{Ca}}^{2+}$-containing stock solutions.²⁷ T-GECO1 was diluted in these buffered solutions, and the fluorescence intensities of the protein in each solution were measured in triplicate. The obtained measurements were plotted on a logarithmic scale against the concentration of free ${\mathrm{Ca}}^{2+}$, and the data were fitted to the Hill equation to determine the apparent $K_{\mathrm{d}}$ and apparent Hill coefficient.

To measure the extinction coefficient, the Strickler–Berg approach was used.²⁸ Briefly, purified T-GECO1 protein was diluted in a ${\mathrm{Ca}}^{2+}$-free buffer (30 mM MOPS, 100 mM KCl, 10 mM EGTA, pH 7.2) and a ${\mathrm{Ca}}^{2+}$ containing buffer (30 mM MOPS, 100 mM KCl, 10 mM Ca-EGTA, pH 7.2). The absorption, fluorescence emission, and excitation spectra for each sample were collected. For fluorescence measurements, the samples were diluted to have optical densities $<0.05$. Excitation spectra in both samples contain only the contribution from the anionic form of the chromophore. Therefore, we calculated the integral of normalized absorption (entering the Strickler–Berg equation) using corresponding excitation spectra. Fluorescence lifetimes and quantum yields (QYs) of the anionic chromophore were measured independently and then used in the Strickler–Berg equation.

Fluorescence lifetimes were measured with a digital frequency domain system ChronosDFD appended to a PC1 spectrofluorimeter (both from ISS, Champaign, Illinois). Fluorescence was excited with a 445-nm laser diode (ISS) through a 440/20 filter. The excitation was modulated with multiple harmonics in the range of 10 to 300 MHz. Coumarin 6 in ethanol with $\tau =2.5\mathrm{ns}$ (ISS) was used as a lifetime standard to obtain the instrumental response function in each measurement. Fluorescence of the sample and standard were collected at 90 deg through a 520LP filter to cut off scattered excitation light. The modulation ratio and phase delay curves were fitted to model functions corresponding to a single- or double-exponential fluorescence decay with Vinci 3 software (ISS). Only double exponential decay functions provided the acceptable ${\chi }^2$ value of 0.5. The main decay component, contributing $\sim 93\%$ of integrated decay in both samples, was used in the Strickler–Berg equation.

Fluorescence QYs were determined using the absolute method with an integrating sphere instrument, Quantaurus-QY (Hamamatsu). In this measurement, the QY was measured as a function of excitation wavelengths between 400 and 500 nm with the step of 5 nm. The QY did not depend on the wavelength in the region from 450 to 475 nm for the ${\mathrm{Ca}}^{2+}$-bound state and from 465 to 480 nm for the ${\mathrm{Ca}}^{2+}$-free state, where the anionic absorption dominated. The average of the QYs in these regions was calculated and is presented in Sec. 3. All measurements were made in triplicate and averaged.

2.4.

Two-Photon Measurements

The two-photon excitation spectra and two-photon absorption cross-sections of T-GECO1 were measured using a previously described protocol.²⁹ Briefly, a tunable femtosecond laser (InSight DeepSee, Spectra-Physics, Santa Clara, California) was coupled to a PC1 Spectrofluorometer (ISS, Champaign, Illinois). The quadratic power dependence of the fluorescence intensity was verified across the spectrum for both proteins and standards. The two-photon cross-section (${\sigma }_2$) of the anionic form of the chromophore was determined for both the ${\mathrm{Ca}}^{2+}$-free and ${\mathrm{Ca}}^{2+}$-bound states, as previously described.³⁰ As a reference standard, a solution of fluorescein in water at pH 12 was used. Fluorescence intensities of the sample and reference were measured for two-photon excitation at 900 nm and for one-photon excitation at 458 nm (${\mathrm{Ar}}^{+}$ laser line). Fluorescence measurements utilized a combination of filters (770SP and 520LP). The two-photon absorption spectra were normalized based on the measured ${\sigma }_2$ values.

2.5.

Kinetic Measurements

Stopped flow kinetic measurements of ${\mathrm{Ca}}^{2+}$ binding and unbinding to T-GECO1 were made using an Applied Photophysics SX20 Stopped-Flow Reaction Analyzer using fluorescence detection. The deadtime of the instrument was 1.1 ms. The mixtures of the protein and ${\mathrm{Ca}}^{2+}$ (or EGTA, for dissociation (or off) rate) were excited at 488 nm with a 2 nm bandwidth, and the emitted light was collected at 515 nm through a 10-mm path. A total of 10,000 data points was collected over three replicates ($n=3$) at increments of 0.01 s for 5 s. For the off rate, T-GECO1 (diluted in $5\mu \mathrm{M}$ ${\mathrm{Ca}}^{2+}$ in TBS) was rapidly mixed 1:1 with 100 mM EGTA (diluted in TBS). Graphpad Prism 9 was used to fit the decrease in fluorescence intensity observed over time to a single exponential dissociation. The $k_{\text{off}}$ determined from this fit is the rate constant for dissociation of ${\mathrm{Ca}}^{2+}$ with units of ${\mathrm{s}}^{-1}$. For the association (or on) rate, T-GECO1 was diluted in a zero free CaEGTA buffer (Thermo Scientific) and mixed 1:1 with varying ${\mathrm{Ca}}^{2+}$ concentrations (150, 225, 351, 602 nM, $1.35\mu \mathrm{M}$). The slope of $k_{\mathrm{obs}}$ versus ${\mathrm{Ca}}^{2+}$ concentration was used to determine the $k_{\text{on}}$ rate (with units of ${\mathrm{s}}^{-1}{\mathrm{M}}^{-1}$).

2.6.

Neuronal Stimulation

T-GECO1, GCaMP6s, and jGCaMP8s were cloned and packaged into an AAV2/1 virus under control of the hSyn promoter. The AAVs were used to transduce the hippocampal and cortical mixture primary cultures from neonatal (P0) pups in poly-D-lysine-coated 24-well glass bottom plates. After 14 days post transduction, the culture medium was exchanged with a 1 mL imaging buffer [145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2 mM ${\mathrm{CaCl}}_2$, 1 mM ${\mathrm{MgCl}}_2$, pH 7.3] containing $10\mu \mathrm{M}$ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), $10\mu \mathrm{M}$ 3-(($R$)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (($R$)-CPP), $10\mu \mathrm{M}$ gabazine, and 1 mM ($S$)-$\alpha$-methyl-4-carboxyphenylglycine (($S$)-MCPG) (Tocris). Neurons were field stimulated with 1, 3, 10, and 20 pulses at 30 Hz and imaged through a 20× objective, with excitation at $470/40\mathrm{nm}$. Brightness data were not corrected for differences in excitation efficiency. Imaging was performed at room temperature.

2.7.

Preparation of Organotypic Hippocampal Slice Cultures for Two-Photon ${\mathrm{Ca}}^{2+}$ Imaging Using T-GECO1 and jGCaMP7s

Organotypic hippocampal slices were prepared from postnatal day 8 (P8) mice (Janvier Labs, C57Bl/6J). Hippocampi were dissected and sectioned into $300\mu \mathrm{m}$ thick slices using a tissue Chopper (McIlwain type 10180, Ted Pella) in a cold dissection medium consisting of GBSS (Sigma, G9779) that was supplemented with 25 mM D-glucose, 10 mM HEPES, 1 mM Na-pyruvate, 0.5 mM $\alpha$-tocopherol, and 20 nM ascorbic acid.

Slices were incubated for 45 min at 4°C in the dissection medium and then placed on a porous membrane (Millipore, Millicell CM PICM03050) and cultured at 37°C, 5% ${\mathrm{CO}}_2$ in a medium consisting of 50% Opti-MEM (Fisher 15392402), 25% heat-inactivated horse serum (Fisher 10368902), 24% HBSS, and 1% penicillin/streptomycin ($5000\mathrm{U}{\mathrm{mL}}^{-1}$) and supplemented with 25 mM D-glucose, 10 mM HEPES, 1 mM Na-Pyruvate, 0.5 mM $\alpha$-tocopherol, and 20 nM ascorbic acid. After 3 days in vitro (DIV), this medium was replaced with one consisting of 82% neurobasal-A (Fisher 11570426), 15% heat-inactivated horse serum (Fisher 10368902), 2% B27 supplement (Fisher, 11530536), 1% penicillin/streptomycin ($5000\mathrm{U}{\mathrm{mL}}^{-1}$), 0.8 mM L-glutamine, 0.8 mM Na-pyruvate, 10 nM ascorbic acid, and 0.5 mM $\alpha$-tocopherol. This medium was removed and replaced every 2 to 3 days. Slices were transduced with AAVs at DIV 3 by bulk application of $1\mu \mathrm{L}$ of virus per slice, for the expression of T-GECO1 or jGCaMP7s under control of the hSyn promoter. Experiments were performed at DIV 10.

2.8.

Two-Photon ${\mathrm{Ca}}^{2+}$ Imaging of Action Potentials in T-GECO1- and jGCaMP7s-Expressing Organotypic Hippocampal Slices

At DIV 10, whole-cell current clamp recordings of T-GECO1- or jGCaMP7s-expressing neurons were performed at room temperature (21 to 23°C). A commercial upright microscope (Zeiss, Axio Examiner.Z1), equipped with a microscope objective (Zeiss, W Plan-Apochromat 20X, 1.0 NA) and an sCMOS camera (Photometrics, Kinetix), was used to collect light transmitted through the sample. Patch-clamp recordings were performed using an amplifier (Molecular Devices, Multiclamp 700B) and a digitizer (Molecular Devices, Digidata 1440A), at a sampling rate of 10 kHz using pCLAMP10 (Molecular Devices). During the experiments, slices were continuously perfused with artificial cerebrospinal fluid (ACSF) composed of 125 mM NaCl, 2.5 mM KCl, 1.5 mM ${\mathrm{CaCl}}_2$, 1 mM ${\mathrm{MgCl}}_2$, 26 mM ${\mathrm{NaHCO}}_3$, 0.3 mM ascorbic acid, 25 mM D-glucose, and 1.25 mM ${\mathrm{NaH}}_2{\mathrm{PO}}_4$. ACSF was supplemented with $1\mu \mathrm{M}$ AP5 (Abcam, ab120003), $1\mu \mathrm{M}$ NBQX (Abcam, ab120046), and $10\mu \mathrm{M}$ picrotoxin (Abcam, 120315). Continuous aeration of the recording solution with 95% ${\mathrm{O}}_2$ and 5% ${\mathrm{CO}}_2$ resulted in a pH of 7.4. Patch pipettes were pulled from borosilicate glass capillaries (with filament, OD: 1.5 mm, ID: 0.86 mm, 10 cm length, fire polished, WPI) using a Sutter Instruments P1000 puller, to a tip resistance of 4.5 to 5.5 MΩ, and filled with an intracellular solution consisting of 135 mM K-gluconate, 4 mM KCl, 4 mM Mg-ATP, 0.3 mM ${\mathrm{Na}}_2$-GTP, 10 mM ${\mathrm{Na}}_2$-phosphocreatine, and 10 mM HEPES (pH 7.35). Only recordings with an access resistance below 20 MΩ were included in the subsequent analysis. In the current-clamp configuration, the bridge potential was corrected (bridge $\text{potential}=13.9\pm 1.0\mathrm{M}\mathrm{\Omega }$; mean ± s.d.).

Two-photon scanning imaging was performed with a Ti:sapphire tunable pulsed laser (Spectra Physics, Mai-Tai DeepSee, pulse $\text{width}\approx 100\mathrm{fs}$, repetition rate 80 MHz, tuning range 690 to 1040 nm), going through a commercial galvo-galvo scanning head (3i, Vivo 2-photon) operated using Slidebook 6 software. The detection axis consisted of a PMT with a 510/84 nm bandpass filter (Semrock, FF01-510/84). Imaging was performed within a $365\times 365\mu \mathrm{m}$ field of view (FOV) at a rate of 3.05 Hz (bidirectional scanning, $256\times 256\text{pixels}$, pixel size $1.4\mu \mathrm{m}$, dwell time $4.0\mu \mathrm{s}$). Laser power was controlled by a Pockels cell (Conoptics, 350-80). Prior to the experiments, power was measured at the output of the objective using a thermal sensor power meter (Thorlabs, PM100D).

Action potentials were triggered by injecting the current for 5 ms (ranging from 500 to 1200 pA) at a rate of 30 Hz during a period ranging from 5 to 650 ms to evoke the desired number of action potentials, while the FOV was scanned under 850 or 920 nm illumination at 20 mW. Recordings were dismissed if the desired amount of action potentials failed to occur.

Fluorescence intensities were integrated over regions of interest (ROIs) covering the patched neuron soma. Percentage changes in fluorescence were calculated as $\mathrm{\Delta }F/F_0=(F-F_0)/F_0$, where $F_0$ is the basal level of fluorescence measured, averaged over 35 frames ($\approx 12\mathrm{s}$) before the triggering of action potentials. SNR was measured as $\mathrm{SNR}=F/{\sigma }_{F0}$, where ${\sigma }_{F0}$ represents the standard deviation of the fluorescence $F$ over the 35 frames prior to the stimulation.

2.9.

Evaluation of Crosstalk Induced by the Two-Photon Scanning Laser in ChroME-Expressing Organotypic Hippocampal Slices

At DIV 3, organotypic hippocampal slices were infected with a mixture of AAV9.hSyn.DIO.ChroME.Flag.ST.P2A.H2B.mRuby3.WPRE.SV40 (titer = 5.9E12 GC ${\mathrm{mL}}^{-1}$) and AAV9.hSyn.Cre.WPRE.hGH (titer = 2.3E11 GC ${\mathrm{mL}}^{-1}$) by bulk application of $1\mu \mathrm{L}$ of the mixture.

At DIV 10, whole-cell current clamp recordings of ChroME-expressing cells were performed in the same conditions as described above. The membrane potential of the patched neuron was monitored and recorded while scanning the FOV for 30 s ($365\times 365{\mu \mathrm{m}}^2$, $256\times 256\text{pixels}$, pixel size $1.4\mu \mathrm{m}$) at 850 or 920 nm, at 20 mW, and at acquisition rates of 1.5, 3.05, and 6 Hz (corresponding to the dwell time per pixel of 6, 4, and $2\mu \mathrm{s}$, respectively). The variation of membrane potential $\mathrm{\Delta }{V}_{\mathrm{m}}$ reported in the paper corresponds to the average of the amplitude of the depolarization peaks induced by the imaging laser during a 30 s scanning epoch. Depolarization peaks were measured as $\mathrm{\Delta }{V}_{\mathrm{m}}=V_{\mathrm{mp}}-V_{\mathrm{m}0}$, where $V_{\mathrm{mp}}$ is the peak of the membrane potential depolarization (one for each frame) and $V_{\mathrm{m}0}$ is the membrane potential of the neuron measured just before the beginning of the scanning. The ratio $\mathrm{\Delta }{V}_{\mathrm{m}850}/\mathrm{\Delta }{V}_{\mathrm{m}920}$ was calculated for each cell and then averaged across cells.

2.10.

Stereotaxic Injection and Fiber Implant Surgery

Stereotaxic injections of AAVs and optical fiber implant surgeries were performed at the same time in C57BL/6J mice (The Jackson Laboratory, #000664) at around P60. Mice were anesthetized with isoflurane and monitored throughout the surgery with tail pinch and breathing rate. First, the skin above the skull was cleaned and removed to allow for the attachment of the headframe and optical fiber implants. Next, a burr hole craniotomy was drilled above the fiber implant coordinates for implantation in the nucleus accumbens core (AP: 1.2 mm, ML: 1.3 mm, DV: 4.1 mm). Virus injection of either AAV2/1-hSyn-T-GECO1 (100 nL, titer = 1.5E13 GC ${\mathrm{mL}}^{-1}$) or AAV2/1-hSyn-jGCaMP8s (100 nL, titer = 1.9E13 GC ${\mathrm{mL}}^{-1}$) was performed with a glass pipette prior to the fiber implant. Following the virus injection, a fiber optic probe was positioned above the same coordinates, and the tip of the fiber was lowered to $100\mu \mathrm{m}$ above the virus injection. The fiber implant was then affixed to the skull with dental cement. A custom headframe was then positioned on the skull and glued in place with dental cement to allow for head-fixation during photometry. The mice were allowed to recover for 2 weeks before the start of imaging. All photometry was performed in head-fixed mice placed on a running wheel to allow for spontaneous running.

2.11.

Fiber Photometry Measurement and Analysis

Fiber photometry measurements were performed on a custom spectral photometry system. 448 nm (Coherent, OBIS 445 nm LX 365 mW LASER, measured wavelength is 448 nm) and 473 nm (Coherent, OBIS 473 nm LX 200 mW LASER, measured wavelength is 473 nm) excitation lasers were co-aligned and focused onto the back pupil of an objective (Nikon, Plan Apochromat, 10×, 0.45 NA, 25 mm FOV). The excitation light was coupled into a fiber optic patch cable (Doric, $200\mu \mathrm{m}$ core, 0.37 NA) by positioning the patch cable at the image plane of the objective. The other end of the patch cable was coupled to the implanted fiber stub. Emitted light from the brain tissue was collected through the same fiber probes and patch cable and passed through a polychromator (Edmund Optics, 50 mm N-SF11 equilateral prism). The polychromator spreads the image of the fiber tip according to its spectrum, which was imaged onto an sCMOS camera sensor (Hamamatsu, Orca Flash 4.0 v3). The excitation lasers and camera sensor were triggered synchronously using an Arduino Teensy board (the excitation lasers were sequentially triggered, whereas the camera sensor was triggered at every frame) at a 24 Hz frame rate. The raw images were acquired and saved through a custom script in the Bonsai reactive programming environment.³¹ The recorded spectra corresponding to either T-GECO1 (485 to 510 nm) or jGCaMP8s (520 to 545 nm) emission were averaged to yield a single intensity time trace. The fractional intensity change was computed by dividing the intensity of each frame by the mean fluorescence of the full trace over time.

2.12.

Animal Care

Animal experiments at Sorbonne Université were conducted in accordance with guidelines from the European Union and institutional guidelines on the care and use of laboratory animals (Council Directive 2010/63/EU of the European Union). Surgery protocol and fiber photometry imaging experiments at the Allen Institute for Neural Dynamics were approved by the Allen Institute Institutional Animal Care and Use Committee (IACUC). Animal experiments at Janelia Research Campus were conducted according to the National Institutes of Health guidelines for animal research and were approved by the Janelia Research Campus Institutional Animal Care and Use Committee and Institutional Biosafety Committee. Procedures in the United States conform to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice were housed under controlled temperature ($\sim 21°\mathrm{C}$) and humidity ($\sim 50\%$) conditions under a reverse light cycle.

3.1.

Development of mTFP1-Based Genetically Encoded ${\mathrm{Ca}}^{2+}$ Indicator, T-GECO1

Our initial template for constructing an mTFP1-based GECI was the mTFP1-based genetically encoded ${\mathrm{Zn}}^{2+}$ indicator, ZnGreen1.²⁵ ZnGreen1 consists of the Zap1 zinc finger inserted into a further engineered version of mTFP1. This version of mTFP1 in ZnGreen1 harbors the nine additional mutations of N42H, N81D, D116G, S146C, T147D, R149K, E168K, R198H, and V218A using mTFP1 numbering (or N42H, N81D, D116G, S323C, T324D, R326K, E345K, R375H, V395A using T-GECO numbering). To construct the initial prototype mTFP1-based GECI, designated T-GECO0.1, we replaced the Zap1 zinc finger of ZnGreen1 with the fused calmodulin (CaM) and CaM-binding peptide (CBP) domain from ncpGCaMP6s.²⁶ The linker sequences from ZnGreen1 were retained. The arrangement of these domains is represented in Fig. 1(a).

Fig. 1

Development and characterization of T-GECO1 as a purified protein. (a) Two views of the modeled structure of the ${\mathrm{Ca}}^{2+}$-bound state of T-GECO1. For the structure representation, mutated residues are shown as magenta sticks, ${\mathrm{Ca}}^{2+}$ is shown as yellow spheres, and the chromophore is shown in space-filling representation. Both the protein structure and the labels are shown in teal for the mTFP1-derived domain, in orange for the CaM domain, in purple for the CaM-binding peptide, and in black for linkers. Colors are consistent with the sequence alignment shown in Fig. 2. The overall structure was predicted using ColabFold.³² The chromophore was positioned using PyMol (Version 2.5.4 Schrödinger, LLC.) to superimpose the structure of mTFP1 (PDB ID 2HQK)²³ with the fluorescent protein portion of the T-GECO1 model. ${\mathrm{Ca}}^{2+}$ ions were similarly positioned by superimposing the CaM domain of GCaMP2 (PDB ID 3EVR)³³ with the CaM portion of the T-GECO1 model. (b) ${\mathrm{Ca}}^{2+}$ titration of T-GECO1. (c) Stopped-flow kinetic measurements of the fluorescence response of T-GECO1 for ${\mathrm{Ca}}^{2+}$ association (left) and dissociation (right). (d) Excitation and emission spectra of T-GECO1 in the presence and absence of ${\mathrm{Ca}}^{2+}$. (e) Two-photon excitation-induced fluorescence of T-GECO1 as a function of wavelength, in the presence and absence of ${\mathrm{Ca}}^{2+}$, with $\mathrm{\Delta }F/F_0$ represented in teal. (f) Two-photon cross-section of T-GECO1 in the ${\mathrm{Ca}}^{2+}$-bound state compared with the two-photon cross-section of EGFP.

As previously described, we define the linkers as additional residues that are inserted between the ${\mathrm{Ca}}^{2+}$-binding domain (CaM fused to CBP) and the gatepost residues 143 and 146 of mTFP1.³⁴ In T-GECO0.1, the linker from the first mTFP1 gatepost (W143) to CaM linker (Linker 1) is Leu–Gly–Asn. Linker 2 from CBP to the second mTFP1 gatepost (S146C) is a single Pro. To develop further improved T-GECO variants, we first optimized these linker residues and some adjacent positions. This was achieved by randomizing each residue, expressing the resulting library in E. coli, picking and culturing bright colonies, and testing ${\mathrm{Ca}}^{2+}$-dependent responses in bacterial lysates. Ultimately, we identified Arg–Asn–Arg as the optimal Linker 1, and Ile as the optimal Linker 2 [Fig. 1(a)].

Further optimization by directed evolution was performed by generating libraries using error-prone (EP) PCR amplification of the entire coding sequence of T-GECO. In each round, we took variants with moderate to high fluorescence change upon binding ${\mathrm{Ca}}^{2+}$ and measured their affinity, pH response, QY, and extinction coefficient. We obtained the DNA sequence of these variants and used them as the template for the next round of iterative directed evolution. Following five generations of screening, we arrived at T-GECO1 on the basis of its high $\mathrm{\Delta }F/F_0$, high affinity, high brightness, two-photon cross-section, and kinetics. T-GECO1 has 25 mutations with respect to T-GECO0.1 (E5D, T96I, H123Y, L144R, G145N, N146R, K175E, E199V, T207A, A218S, K222N, R251H, H252R, T262S, E268D, M269V, Q280L, R304H, P322I, C323G, D324G, K339E, K341E, T400R, and D401R, using T-GECO numbering). There are four mutations in the linkers, nine mutations in the mTFP1-derived region, 11 mutations in CaM, and 1 mutation in CBP. The locations of all mutations are shown in Figs. 1(a) and 2.

Fig. 2

Sequence alignment of T-GECO1 and related proteins. Residues are colored teal for the mTFP1-derived domain, orange for the CaM domain, purple for the CaM-binding peptide, and black on a gray background for the linkers. Mutated residues are shown on a magenta background. A black box encloses the chromophore-forming tripeptide. The gatepost residues 143 and 146 (using mTFP1 numbering) are underlined.³⁴ Colors are consistent with the structural model shown in Fig. 1(a).

We first characterized the photophysical properties of T-GECO1 as a soluble protein under one-photon and two-photon excitation [Figs. 1(b)–1(f)]. Under one-photon excitation, T-GECO1 in the ${\mathrm{Ca}}^{2+}$-bound state exhibits excitation and emission peaks at 468 and 500 nm, respectively. The molecular brightness of T-GECO1 in the ${\mathrm{Ca}}^{2+}$-bound state, calculated as the product of the extinction coefficient ($\mathrm{49,300}{\mathrm{M}}^{-1}{\mathrm{cm}}^{-1}$) and QY (0.83), is similar to that of EGFP (Table 1).¹⁷ The two-photon excitation maximum of T-GECO1 is 888 nm with a brightness of 83 GM, which is 1.4× the value of mTFP1 and 2× the value of EGFP (Table 2). T-GECO1 exhibits a large change in fluorescence intensity upon the addition of ${\mathrm{Ca}}^{2+}$, with 1-photon peak $\mathrm{\Delta }F/F_0$ of 15 and 2-photon peak $\mathrm{\Delta }F/F_0$ of 7, where $\mathrm{\Delta }F/F_0=(F_{\max }-F_{\min })/F_{\min }$. In addition, we determined that T-GECO1 has an apparent $K_{\mathrm{d}}$ of 82 nM for binding to ${\mathrm{Ca}}^{2+}$ and an apparent Hill coefficient ($n_H$) of 3.6. T-GECO1 exhibits moderate binding (on) and dissociation (off) kinetics as a soluble protein, with $k_{\text{on}}$ of $8.5\times {10}^5{\mathrm{M}}^{-1}{\mathrm{s}}^{-1}$ and $k_{\text{off}}$ of $1.02{\mathrm{s}}^{-1}$.

Table 1

One-photon photophysical properties of T-GECO1 and mTFP1,28 measured as purified proteins (n=3, averaged). Extinction coefficients were obtained using Strickler–Berg formula.28 In this calculation, the main fluorescence lifetime component of T-GECO1 was used. Note that the relative values of the brightness of the Ca2+-bound and Ca2+-free fluorescent states shown here do not represent the Ca2+-dependent fluorescence change of T-GECO1. The Ca2+-dependent fluorescence change is primarily due to a change in the protonation state of the chromophore, which changes the fraction of the protein in the fluorescent state.

Table 2

Two-photon photophysical properties of T-GECO1 (Ca2+-bound state), mTFP1, and EGFP, measured as purified proteins (n=3, averaged). Values for mTFP1 and EGFP were previously reported.17,28

Together, these results demonstrate that T-GECO1 has favorable photophysical characteristics that make it a potentially useful new GECI. Its large fluorescence change upon binding ${\mathrm{Ca}}^{2+}$, high brightness and two-photon cross-section, and reasonable association and dissociation kinetics suggest that T-GECO1 is a promising tool for monitoring ${\mathrm{Ca}}^{2+}$ dynamics using blue-shifted excitation.

3.2.

Imaging of ${\mathrm{Ca}}^{2+}$ in Electric Field Stimulated Neuronal Cultures

To characterize T-GECO1 in neuronal cultures using one-photon excitation (excitation at 450 to 490 nm), we expressed it under the control of human synaptic (hSyn) promoter in rat primary cortical and hippocampal neurons. We compared the performance of T-GECO1 with GCaMP6s and jGCaMP8s [Fig. 3(a)]. To evoke neuronal activity, we applied trains of 1, 3, 10, and 20 electric field stimuli (FS) and analyzed the resulting fluorescence changes [Figs. 3(b)–3(d)]. T-GECO1 exhibited a peak change in fluorescence ($\mathrm{\Delta }F/F_0$) of 3% for a single stimulus. In comparison, GCaMP6s and jGCaMP8s had peak responses of 9% and 20%, respectively, in response to single stimuli. T-GECO1 exhibited lower $\mathrm{\Delta }F/F_0$ values across all numbers of stimuli tested. The baseline brightness of T-GECO1 ($536\pm 59\mathrm{RFU}$) was found to be 16% higher than that of GCaMP6s ($463\pm 16\mathrm{RFU}$) and 15% higher than that of jGCaMP8s ($466\pm 21\mathrm{RFU}$) [Fig. 3(e)]. Following continuous exposure for 15 s, T-GECO1 maintained $91.7\pm 0.1\%$ of its initial fluorescence intensity, whereas GCaMP6s and jGCaMP8s maintained $>99\%$. T-GECO1 exhibited a marginally larger SNR compared with GCaMP6s and jGCaMP8s, partially due to its higher baseline brightness [Fig. 3(f)].

Fig. 3

Characterization of T-GECO1 in rat cultured neurons. (a) Images of primary rat hippocampal cultured neurons expressing GCaMP6s, jGCaMP8s, and T-GECO1 under the hSyn promoter at baseline and after field stimulations of 1 and 20 field stimulation (FS) pulses at room temperature. Scale bar, $100\mu \mathrm{m}$. (b) Normalized $\mathrm{\Delta }F/F_0$ traces for stimulations at 1 field stimulus and (c) 3 FS, 10 FS, and 20 FS at 30 Hz. (d) Peak $\mathrm{\Delta }F/F_0$ of the three sensors across the same conditions. (e) Baseline brightness of the three sensors. (f) SNR for the three variants across conditions. Traces and error bars denote mean ± s.e.m. Each data point is one ROI and is pooled across three independent wells.

These results demonstrate that T-GECO1 has sufficient sensitivity for detecting small numbers of field stimulation pulses in cultures using one-photon excitation. However, further optimization will be necessary to achieve the peak sensitivity exhibited by late-generation GCaMP series indicators. Nevertheless, T-GECO1’s higher baseline brightness and blue-shifted excitation and emission may prove advantageous, relative to the GCaMP series, for certain one-photon excitation applications such as multicolor imaging and combined use with longer-wavelength activatable optogenetic tools.

3.3.

Two-Photon ${\mathrm{Ca}}^{2+}$ Imaging of T-GECO1 in Organotypic Hippocampal Slices

Next, we compared the performance of T-GECO1 with jGCaMP7s (Ref. 3) using two-photon ${\mathrm{Ca}}^{2+}$ imaging in neonatal mouse organotypic hippocampal slices [Fig. 4(a)]. We hypothesized that, due to its blue-shifted two-photon excitation maxima relative to GCaMP7s’s [Fig. 1(f)], T-GECO1 is a more suitable choice for all-optical stimulation and imaging when used in conjunction with the ChroME opsin.³⁵ Specifically, we expected that the excitation wavelengths that are near-optimal for T-GECO1 (i.e., $\sim 850\mathrm{nm}$) would result in less undesirable activation of ChroME than excitation wavelengths that are near-optimal for GCaMP7s (i.e., $\sim 920\mathrm{nm}$). To test this hypothesis, we quantified the change in membrane potential when ChroME-expressing neurons were illuminated with either 850 or 920 nm and expressed the ratio calculated as $\mathrm{\Delta }{V}_{\mathrm{m}850}/\mathrm{\Delta }{V}_{\mathrm{m}920}$, on a cell-by-cell basis. Across all tested frequencies (1.5, 3, and 6 Hz), this ratio consistently remained below one (0.62, 0.73, 0.67), indicating that using an imaging wavelength of 850 nm rather than 920 nm is advantageous for reducing undesirable ChroME activation [Figs. 4(b) and 4(c)].

Fig. 4

Two-photon ${\mathrm{Ca}}^{2+}$ imaging of T-GECO1 in organotypic hippocampal slices. (a) Schematic of the setup. PC = pockels cell, GM = galvo mirrors, and DM = dichroic mirror. (b) $\mathrm{\Delta }{V}_{\mathrm{m}850}/\mathrm{\Delta }{V}_{\mathrm{m}920}$ ratio of ChroME-expressing organotypic hippocampal slices. (c) $\mathrm{\Delta }{V}_{\mathrm{m}}$ for ChroME-expressing organotypic hippocampal slices induced by 850 or 920 nm laser illumination at 3 Hz. (d) Representative traces of T-GECO1 (teal) and jGCaMP7s (purple) for 1 action potential (top) and 20 action potentials (bottom) excited at 850 nm. (e) Baseline brightness (A.U) for the two indicators at baseline (before stimulation) and at peak (maximum brightness after stimulation) at 850 nm excitation. (f) Normalized fluorescence decay for the two indicators at 850 nm excitation. (g) Peak $\mathrm{\Delta }F/F_0$ (%) for the two indicators at 1 or 20 action potentials at 850 nm excitation. (h) SNR for the two indicators with respect to action potentials at 850 nm excitation. (i) Representative traces of T-GECO1 (teal) and jGCaMP7s (purple) for 1 action potential (top) and 20 action potentials (bottom) excited at 920 nm. (j) Baseline brightness (A.U) for the two indicators at baseline (before stimulation) and at peak (maximum brightness after stimulation) at 920 nm excitation. (k) Normalized fluorescence decay for the two indicators at 920 nm excitation. (l) Peak $\mathrm{\Delta }F/F_0$ (%) for the two indicators at 1 or 20 action potentials at 920 nm excitation. (m) SNR for the two indicators with respect to action potentials at 920 nm excitation. Error bars denote ± S.E.M.

We next investigated the fluorescence responses and rates of fluorescence decay to baseline following stimulated action potentials (APs) of both T-GECO1 and GCaMP7s at excitation wavelengths of 850 and 920 nm with varying numbers of APs [Fig. 4(a)]. Under excitation at 850 nm, T-GECO1 exhibited a fluorescence change ($\mathrm{\Delta }F/F_0$) of 73%, whereas jGCaMP7s exhibited a change of 13%, in response to 1 AP [Figs. 4(d) and 4(g)]. In response to 20 APs, T-GECO1 exhibited a fluorescence change of 450%, and jGCaMP7s exhibited a change of 38% [Figs. 4(d) and 4(g)]. T-GECO1 exhibited lower baseline and peak brightness levels compared with jGCaMP7s when excited at 850 nm (baseline fluorescence of 50.9 AU for T-GECO1 and 142.1 AU for jGCaMP7s; peak fluorescence of 81.6 AU for T-GECO1 and 157.0 AU for jGCaMP7s). The fluorescence decay for T-GECO1 was faster ($t_{1/2}=1.32\mathrm{s}$) than jGCaMP7s ($t_{1/2}=3.98\mathrm{s}$) under 850 nm illumination [Fig. 4(f)]. The signal-to-noise ratio (SNR) for T-GECO1 was substantially higher than for jGCaMP7s [Fig. 4(h)].

When excited at 920 nm, the differences between the two indicators were marginal for 1 AP, with T-GECO1 and jGCaMP7s displaying similar $\mathrm{\Delta }F/F_0$ values (65% and 75%, respectively). For 20 APs, T-GECO1 exhibited a $\mathrm{\Delta }F/F_0$ of 547%, $\sim 2.8$ times greater than the $\mathrm{\Delta }F/F_0$ of jGCaMP7s (194%) [Figs. 4(i) and 4(l)]. The baseline brightness of T-GECO1 before stimulation was higher than that of jGCaMP7s (53.6 AU for T-GECO1 and 37.5 AU for jGCaMP7s) and remained higher at its peak after stimulation (88.1 AU and 79.9 AU, respectively) [Fig. 4(j)]. Similar to the 850 nm excitation, the fluorescence decay for T-GECO1 was faster ($t_{1/2}=1.00\mathrm{s}$) than jGCaMP7s ($t_{1/2}=2.38\mathrm{s}$) under 920 nm illumination [Fig. 4(k)]. There was no noticeable photobleaching observed during the 30 s recordings for jGCaMP7s and T-GECO1 with either 850 or 920 nm excitation. The SNR of T-GECO1 was consistently higher than that of jGCaMP7s [Fig. 4(m)].

These results indicate that T-GECO1 may offer substantial performance advantages relative to jGCaMP7s under two-photon excitation conditions, particularly at the excitation wavelength of 850 nm. This apparent advantage is consistent with our original rationale for using mTFP1, which is itself particularly bright under two-photon excitation as the starting point for developing a new ${\mathrm{Ca}}^{2+}$ indicator.

3.4.

In Vivo ${\mathrm{Ca}}^{2+}$ Detection in the Nucleus Accumbens Using Fiber Photometry

To evaluate the performance of T-GECO1 in the intact brain using one-photon excitation, we conducted fiber photometry measurements by expressing either T-GECO1 or jGCaMP8s in the nucleus accumbens of mice. Fluorescence traces were recorded using fiber implants positioned above the injection site [Figs. 5(a) and 5(b)]. We excited both T-GECO1 and jGCaMP8s using either 448 or 473 nm wavelength while the mice engaged in spontaneous running, with occasional manual whisker flicking to evoke ${\mathrm{Ca}}^{2+}$ transients. T-GECO1 enabled the reliable detection of ${\mathrm{Ca}}^{2+}$ transients at both 473 and 448 nm excitation wavelengths, with higher fluorescence changes ($\mathrm{\Delta }F/F_0$) observed at 473 nm compared with 448 nm [Figs. 5(c) and 5(d)]. In general, these responses were substantially lower than those observed with jGCaMP8s, regardless of the excitation wavelength used [Figs. 5(e) and 5(f)].

Fig. 5

In vivo ${\mathrm{Ca}}^{2+}$ detection in the nucleus accumbens using fiber photometry. (a) Simplified schematic illustrating the fiber photometry setup, featuring two excitation wavelengths of 473 and 448 nm. (b) Precise position of the fiber optic implant. (c) Representative fluorescence traces of T-GECO1 at 473 nm excitation (left) and 448 nm excitation (right). (d) Zoomed-in view of the outlined traces displayed in (c). (e) Representative fluorescence traces of jGCaMP8s at 473 nm excitation (left) and 448 nm excitation (right). (f) Zoomed-in view of the outlined traces displayed in (e).

These in vivo imaging results are qualitatively consistent with the results from in vitro imaging in neuronal cultures using one-photon excitation, that is, T-GECO1 can be effectively utilized for in vivo one-photon excitation imaging of neuronal activity using either 448 or 473 nm excitation, but it does not achieve the peak sensitivity exhibited by late-generation GCaMP series indicators.