(a) Screening resulted in several mutants with improved baseline brightness and signal change in HEK293 cells. (b) Schematic representation of GCaMP2 and GCaMP3. Mutated residues are highlighted in red. (c) Fluorescence spectra of GCaMP3 and GCaMP2 (1 μM protein) with 1 mM Ca²⁺ or 10 mM EGTA in MOPS buffer (30 mM MOPS, 100 mM KCl, pH 7.5) (average of three independent measurements). The fluorescence intensity of each indicator was normalized to the peak of the calcium-saturated spectrum. The inset shows the un-normalized fluorescence emission spectra (485 nm excitation). (d) Ca²⁺ titration curve (1 μM protein) in MOPS buffer. Inset shows the dynamic range of the two indicators. (e) The improved baseline fluorescence of GCaMP3 compared to GCaMP2. Both indicators were either transfected into HEK293 cells or virally delivered to layer 2/3 cortical neurons. Images were taken either 48 hours post-transfection or 12 days post-viral injection, then analyzed with Volocity 5.0 (Improvision). 50 μm scale bar. Error bars indicate standard deviation of the mean.

(a) Schematic representation of the FRET-based calcium indicators D3cpV and TN-XXL. (b) Mean of fluorescence responses for action potential trains across cells (n = 7 cells, 1 trial each cell). Traces from bottom to top represent the response to trains of 1, 2, 3, 5, 10, 20 and 40 APs. (c) Ratio change of D3cpV and TN-XXL for individual hippocampal pyramidal cells (thin gray lines) in response to trains of action potentials given at 83 Hz, and the mean across cells (thick black line). (d) SNR of D3cpV and TN-XXL. (e, f) Comparison of mean responses (ΔF/F or ΔR/R) and SNR of GCaMP3, D3cpV and TN-XXL. Zoom of lower stimuli is shown in inset. (g) Mean cellular fluorescence during periodic two-photon frame scans (n = 3–4 cells per GECI). (h) Rise and decay time comparison of all three indicators at 10 APs. Error bars indicate standard deviation of the mean.

Odour-evoked responses of GCaMP1.6 and GCaMP3 in projection neurons of the Drosophila antennal lobe (AL). (a) Expression of GCaMP1.6 and GCaMP3 in DM2 glomeruli of the AL. DM2 ROI, circled with dashed line, was used for frame-scans. 10 μm scale bar. (b) Comparison of DM2 frame-scan responses of GCaMP1.6 and GCaMP3 to presentations of vinegar. 5-trial average response of a single animal each, expressing GCaMP1.6 (left panel) or GCaMP3 (right panel). (c) Peak response of GCaMP1.6 (4ALs from 3 animals) and GCaMP3 (4ALs from 4 animals) across all trials and animals. The response of GCaMP3 was increased ~4-fold compared to GCaMP1.6. Comparisons shown here are significant (p = 6.80e-08, Mann-Whitney). Error bars indicate standard deviation of the mean.

(a) Line-scan location at the base of the apical dendrite and evoked action potentials in the soma. 10 μm scale bar. Raw line-scan images showing fluorescence baseline and single action potential-evoked responses. (b) Average-trial responses of GCaMP3 for individual hippocampal pyramidal cells in organotypic slices (n = 9 cells, thin gray lines) and mean across all cells (thick black line) for each stimulus. Note different y-axis scales for each panel. (c) Expression of GCaMP3 in layer 2/3 cortical neurons (S1) via in utero electroporation. 20 μm scale bar. (d) Average-trial responses of GCaMP3 for individual layer 2/3 cortical cells (n = 9 cells, thin gray lines) in response to trains of action potentials given at 83 Hz, and the mean across cells (thick black line). Note different y-axis scales for each panel. (e, f) Amplitudes and SNR of GCaMP3 responses for individual hippocampal pyramidal cells (thin gray lines) in response to trains of action potentials given at 83 Hz, and the mean across cells (thick red line). (g) The average response of GCaMP3 is greater than GCaMP2. (h) The SNR of GCaMP3 is also greater than GCaMP2. Error bars indicate standard deviation of the mean.

Odour-evoked responses of GCaMP1, GCaMP2 and GCaMP3 in C. elegans olfactory neurons. Transgenic worm lines expressing GCaMPs were imaged following an odour addition-removal sequence. (a,b) Upon odour presentation, GCaMP3 and GCaMP2 showed a similar decrease in fluorescence intensity. (c,d) Upon odour removal, GCaMP3 showed a 4- to 5-fold increase of fluorescence response compared to GCaMP2 and GCaMP1. Grey bars denote odour presence. Yellow intervals were analyzed in b and d. Grey shading of each trace and error bars indicate standard error of the mean (S.E.M., n = 12 animals for each genotype).

(a) Schematic illustrating simultaneous two-photon imaging and electrophysiology in virally infected L2/3 neurons in vivo. (b) Examples of single-trial responses (gray line) and average across 10 trials (black line) from three neurons to evoked APs at 50 Hz under anesthesia. (c) GCaMP3 showed linear ΔF/F in response to evoked APs (n = 9 cells, thin gray lines; average of 10 trials per neuron, thick red line). (d) Example trace of simultaneous recording of fluorescence changes (black) and spiking activity (red, number of APs per 0.5 sec bin) during head-fixed behavior. APs were recorded in loose seal cell-attached mode. (e) Fluorescence change in response to action potentials, binned over 0.5 s intervals. 6 cells from 3 animals were indicated with different colors. Error bars indicate standard deviation of the mean. (f) Cumulative distribution of the decay times (T_½, single-exponential fit from last fluorescence maximum). Decay times of neurons with nuclear exclusion are similar at 10 to 120 days (colored lines, p = 0.22, Kolmogorov–Smirnov test). Nuclear-filled neurons have significantly longer decay times (black line, p = 5.78e-10, Kolmogorov–Smirnov test). (g) GCaMP3 expression in L2/3 neurons of the primary motor cortex at 72 days post injection (top, 30 μm scale bar) and ΔF/F traces of individual cells (bottom, black lines). Relative treadmill movement indicated by red line (F: forward, B: backward). (h) The same field of view and fluorescent traces as (g) at 120 days post injection.