Far-red fluorescent genetically encoded calcium ion indicators

Abstract

Genetically encoded calcium ion (Ca²⁺) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca²⁺ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm, respectively, display large responses to Ca²⁺ in vitro (ΔF/F₀ = 6 for FR-GECO1a, 18 for FR-GECO1c), are bright under both one-photon and two-photon illumination, and have high affinities (apparent K_d = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca²⁺. FR-GECOs offer sensitive and fast detection of single action potentials in neurons, and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators.

Fig. 1. Engineering and characterization of FR-GECO1a and FR-GECO1c.

A Selected far-red FPs and FR-GECO genealogy. Far-red FPs (λ_em > 620 nm) are highlighted in red. B Schematic illustration of FR-GECO1 design and engineering. Linker residues are in grey; asterisk indicates position 35. Magenta and blue-colored amino acids (and correspondingly colored circles in the schematic) represent the positions of ‘gatepost’ residues that define the gatepost residues of cpmKelly2. Abbreviations: Calmodulin (CaM); CaM-dependent kinase kinase CaMKK-α/β peptide (ckkap). Absorbance, excitation, and emission spectra of FR-GECO1a (C) and FR-GECO1c (D) with dashed lines representing the fluorescence spectra of the Ca²⁺-free state and solid lines representing the spectra of the Ca²⁺-bound state. Two-photon spectra and dynamic range of FR-GECO1a (E) and FR-GECO1c (F). K_d titration (n = 3 technical replicates) with FR-GECO1a (G) and FR-GECO1c (H). pH sensitivity (n = 3 technical replicates) of FR-GECO1a (I) and FR-GECO1c (J). Error bars represent S.E.M. Source data are provided in a Source Data file.

Fig. 2. Performance of FR-GECO1 variants in cultured neurons.

A Representative images of neurons expressing jRGECO1a (repeated in 96 wells over 8 plates), jRCaMP1a (repeated in 77 wells over 8 plates), FR-GECO1a (repeated in 58 wells over 5 plates), FR-GECO1c (repeated in 47 wells over 5 plates), at baseline, and stimulation with 3 action potentials (AP), 10 action potentials, and 160 action potentials. All repeats gave similar results. Scale bar is 200 μm in all images. B, C Fluorescence response as a function of time following 3 action potentials (B) and 10 action potentials (C). Fluorescence responses (D), half rise time (E), half decay time (F), SNR (G), and basal fluorescence (H), of jRGECO1a (black; n = 1482 cells), jRCaMP1a (green; n = 1043 cells), FR-GECO1a (red; n = 601 cells), and FR-GECO1c (blue; n = 171 cells). Colors are consistent throughout the figure. All indicators were excited with 565 nm LED and imaged with mCherry filter cubes (excitation 560/40 nm, dichroic mirror 585LP and emission 630/75 nm). For D–G, error bars represent S.E.M. For H, the box indicates the 25th to 75th percentile range, the horizontal line indicates the median, and the whiskers extend from the box to the farthest data point lying within 1.5× the inter-quartile range (IQR). Data points beyond the boundary of the higher whisker were removed.

Fig. 3. Comparison between FR-GECO1 and near-infrared GECIs in the hearts of embryonic zebrafish in vivo^–.

Fluorescence responses (A), average intensities (AU arbitrary units) (C), and SNRs (D) of FR-GECO1a, FR-GECO1c, NIR-GECO2, NIR-GECO2G, and iGECI. B Average of 5 spontaneous heartbeats from representative traces for each sensor (left) and representative traces (right) of FR-GECO1a and FR-GECO1c. Colors are consistent with other panels and iGECI was omitted for A and B since no Ca²⁺-dependent change in fluorescence was observed. A–D n = 23 embryos for FR-GECO1a, 23 embryos for FR-GECO1c, 25 embryos for NIR-GECO2, 25 embryos for NIR-GECO2G, and 12 embryos for iGECI. Error bars are standard deviation. Statistical comparisons were performed using two-sided Welch’s t-test with Benjamini–Hochberg multiple test correction (Shapiro–Wilk test for normality p > 0.5 for all groups); n.s. not significant, *** p < 0.001, **** p < 0.0001. Exact p values are: A FR-GECO1a vs. FR-GECO1c p = 8.526e−4, FR-GECO1a vs. NIR-GECO2 p = 1.321e−5, FR-GECO1a vs. NIR-GECO2G p = 8.468e−6, FR-GECO1c vs. NIR-GECO2 p = 1.7e−5, FR-GECO1c vs. NIR-GECO2G p = 1.594e−5, NIR-GECO2 vs. NIR-GECO2G p = 0.8796; C FR-GECO1a vs. FR-GECO1c p = 2.474e−5, FR-GECO1a vs. NIR-GECO2 p = 0.3249, FR-GECO1a vs. NIR-GECO2G p = 7.260e−2, FR-GECO1c vs. NIR-GECO2 p = 6.933e−10, FR-GECO1c vs. NIR-GECO2G p = 1.71e−4, NIR-GECO2 vs. NIR-GECO2G p = 8.399e−4; D FR-GECO1a vs. FR-GECO1c p = 0.1057, FR-GECO1a vs. NIR-GECO2 p = 2.227e−4, FR-GECO1a vs. NIR-GECO2G p = 2.761e−4, FR-GECO1c vs. NIR-GECO2 p = 5.785e−5, FR-GECO1c vs. NIR-GECO2G p = 6.629e−5, NIR-GECO2 vs. NIR-GECO2G p = 0.6697. Source data are provided in a Source Data file.

Fig. 4. Ca²⁺ optopatch for all-optical electrophysiology in cultured neurons and in zebrafish embryonic heart in vivo.

A Average fluorescence trace of cultured rat hippocampal neurons expressing FR-GECO1a or 1c via transient transfection, under constant 561 nm illumination (13 mW/mm²) and 100 ms flash of 470 nm LED illumination (23 mW/mm²). Time-average of fluorescence increase induced by 470 nm illumination for each neuron was quantified and shown in red (FR-GECO1a) or blue (FR-GECO1c) dots (n = 6 for both FR-GECO1a and 1c). B HEK293T cells transiently transfected with CheRiff were illuminated by alternating pulses of 488 nm and 594 nm light of increasing intensities (top). Photocurrents were measured under a voltage clamp at −65 mV (middle). Photocurrents at 594 nm were then normalized to the maximum photocurrent induced by excitation with 488 nm (bottom). Typical light intensities used for optopatch experiments are highlighted in orange (n = 3 cells). Average fluorescence traces of cultured rat hippocampal neurons expressing FR-GECO1a (or 1c)-P2A-CheRiff-eGFP-Kv2.1 (AAV transduction) under constant 594 nm illumination (10 mW/mm²) and 470 nm optical stimulation for 0.6 s at 0.07 mW/mm² (C) or 0.35 mW/mm² (D) (n = 45 neurons for FR-GECO1a; n = 37 neurons for FR-GECO1c). Blue light crosstalk during the stimulation epochs (dotted lines) has been subtracted from the traces. Error bands represent S.E.M. E Isochronal map of calcium wave in an embryonic zebrafish linear heart tube (LHT) at 24 h post-fertilization (hpf). The map was calculated from a spike-triggered average (n = 12 beats). Each isochron is 50 ms, propagating from left to right. Cardiac marker Nkx2.5:ZsYellow marked in green. Scale bar 50 μm. F Mean FR-GECO1c ΔF/F of the LHT in (E). Simultaneous pacing and measurement of Ca²⁺ dynamics in the LHT using FR-GECO1a combined with optical stimulation at 1 Hz using CheRiff (G) and CoChR (H). FR-GECOs were imaged using constant 594 nm illumination at 24–290 mW/mm². Channelrhodopsin was excited using 488 nm light at 24 mW/mm² (25 ms pulse for CoChR, 75 ms pulse for CheRiff). All images were acquired at 33 Hz. Source data are provided in a Source Data file.