ASAP4e

Speed-optimized ASAP4 variant with fast kinetics and a large fluorescence increase per action potential.

Category: VSD-cpGFP GEVI
Published: 2023-07-06 — Nature Methods 2023
Derived from: ASAP3
Response polarity: positive-going (fluorescence increases on depolarization)
Spectrum: excitation peak 375 nm, emission peak 524 nm (ASAP4e)
Two-photon compatible: yes
Plasmid: Addgene #201031
Tags: cpGFP, Positive-going, Two-photon

Headline values (as shown in the GEVIBench table)

Metric	Value	Definition
Brightness (B/B_EGFP)	0.17× EGFP	Molecular brightness relative to EGFP (extinction coefficient × quantum yield, or a measured ratio). Sensors not measured against EGFP directly are placed through a chain of pairwise brightness comparisons, combined by geometric mean.
Kinetics τ_on / τ_off (ms)	6.3 / 5.7 ms	Activation / decay time constants in ms (amplitude-weighted mean τ for multi-exponential fits). The value shown averages the measurements made at physiological temperature (≥33 °C); if there are none, it is the measurement closest to 33 °C.
ΔF/F per 100 mV	+210% (median of 3 measurements: +211%)	Steady-state fluorescence change for a 100 mV depolarization step (−70 → +30 mV); the sign gives the polarity. The table shows the first listed measurement; the median across measurements (one-photon preferred) is used for sorting.
ΔF/F per mV	1.1%	Subthreshold sensitivity: fluorescence change per mV near rest (−90 to −50 mV). Where no value is reported directly, it is estimated from the F–V curve.
ΔF/F per AP	38% (median of 5 measurements: 33%)	Fluorescence change per single action potential in neurons. The table shows the first listed measurement; the median across measurements (one-photon preferred) is used for sorting.
AP width (FWHM, ms)	5 ms	Full width at half maximum of the optical spike waveform.
Photostability t75% @ 100 mW/mm²	—	Time for fluorescence to fall to 75% of its initial value under one-photon illumination, linearly dose-scaled to 100 mW/mm² (t50% is used where the 75% crossing falls inside a fast initial transient). Two-photon curves are not dose-scaled and are excluded.
Papers using it (N_used)	6	Number of published research papers, tracked by GEVIBench, that used the sensor for an experiment (a popularity / track-record measure).

Measurements and sources

Kinetics

τ_on 18.29 ms, τ_off 15.6 ms at 22°C — Evans 2023, Table S1
τ_on 4.55 ms, τ_off 4.71 ms at 37°C — Evans 2023, Table S1
τ_on 8.14 ms, τ_off 6.7 ms at 33°C — McDonald 2026, Table S1.4

ΔF/F per 100 mV

+210%, 1P — Evans 2023, Table 1
+148%, 2P — McDonald 2026, Fig. 1F
+212%, 1P — Lee 2024, Table S1

ΔF/F per mV (subthreshold)

1.12%/mV, 2P (Cross-measured in the FORCE1s paper (McDonald et al. 2026). Subthreshold steady-state response to a 30-mV depolarization from −70 → −40 mV = 33.6 ± 6.4% (n = 5, whole-cell voltage clamp + 2P imaging at 33 °C, HEK293A; main text and Fig. 1E/Fig. S1.2C). Expressed as a slope: 33.6% / 30 mV ≈ 1.12 %/mV (cf. FORCE1s 87.4% → 2.91 %/mV, JEDI-2P 25.9% → 0.86 %/mV over the same step).) — McDonald 2026, Fig. 1E

ΔF/F per action potential

AP width (FWHM)

5.8 ms at 400 Hz sampling, Mouse barrel cortex L2/3 pyramidal neurons, in vivo, whisker stimulation (ASAP4e-Kv), 2P, 37°C — Lee 2024, Fig. 3d
4.2 ms at 721 Hz sampling, HEK293A cells, whole-cell voltage clamp, in vitro (2-ms FWHM command AP waveform), 2P, 33°C — McDonald 2026, Fig. 1K

Brightness comparisons

0.362× the brightness of ASAP3 — Evans 2023, Ext. Fig. 3d
0.5× the brightness of FORCE1s — McDonald 2026, Fig. 1C
0.6666666666666666× the brightness of ASAP5 — Lee 2024, Table S4
1× the brightness of ASAP6c — Lee 2024, Table S4
0.03571428571428571× the brightness of pAce — Lee 2024, Table S4
0.2857142857142857× the brightness of Positron — Lee 2024, Table S4

Photobleaching

t75% = 104 s at 100 mW at sample plane (2P, 940 nm), 2P — McDonald 2026, Fig. S1.2B
curve only (did not reach 75% within the measured window) at 100 mW/mm², 1P — Evans 2023, Fig. 2b
t75% = 141 s at 960 mW/mm² (2P galvanometric scanning, 5 min), 2P — Evans 2023, Ext. Fig. 4c

Fluorescence–voltage (F–V) curve

ΔF/F (%) at each membrane potential, normalized to 0 at −70 mV: -120 mV: -13%; -100 mV: -9%; -80 mV: -3%; -70 mV: 0%; -60 mV: 6%; -40 mV: 22%; -20 mV: 55%; 0 mV: 98%; 30 mV: 210%; 50 mV: 300%; 70 mV: 360%; 90 mV: 410%; 120 mV: 460% — Evans 2023, Fig. 1B

Research papers using ASAP4e (6)

A positively tuned voltage indicator for extended electrical recordings in the brain — Evans SW, Shi DQ, Chavarha M, et al., Nature Methods 2023 (HEK293A cells (voltage clamp), mouse hippocampus and cortex, in vivo (origin paper introducing ASAP4e))
Improving positively tuned voltage indicators for faster kinetics and higher contrast — Lee S, Zhang G, Hao YA, et al., bioRxiv 2024 (HEK293A cells, voltage clamp and AP-waveform characterization of ASAP4e)
A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo — McDonald AJ, Land MA, Yang S, et al., bioRxiv 2026 (HEK293A cells, ASAP4e benchmarked under 2P excitation against FORCE1s (kinetics, F-V, brightness, spike waveforms))
Voltage imaging reveals hippocampal inhibitory dynamics shaping pyramidal memory-encoding sequences — Taxidis J, et al., Nature Neuroscience 2025 (Mouse, hippocampus CA1, PV and SST interneurons, in vivo)
Imaging sensory transmission and neuronal plasticity in primary sensory neurons with a positively tuned voltage indicator — Zhang Y, et al., Nature Communications 2025 (Mouse, dorsal root ganglia neurons, in vivo)
High-speed neural imaging with multiplexed miniaturized two-photon microscopy — Zhang Z, Liu SJ, Mattison B, Muir J, Spurr N, Kim CK, Yang W, Cell Reports Methods 2025 (Mouse, primary visual cortex (V1), freely moving, in vivo; two-photon voltage imaging of ASAP4e at 400 Hz over a 380 x 150 um FOV at 140-280 um depth (M-MINI2P))

Data and citation

llms.txt — summary for language models
llms-full.txt — every sensor in full (Markdown)
gevis.json — all values and measurements with sources (JSON)
gevis.csv — one row per sensor (CSV)

Cite GEVIBench as: Adam E. Cohen and Daozheng Gong (2026). Voltage imaging: A practical guide. Neuron. https://doi.org/10.1016/j.neuron.2026.08.002

Generated from the GEVIBench database on 2026-10-12.