ASAP Voltage Indicator
A protein that lives in a neuron's membrane and flashes brighter every time the cell fires — turning thought into visible light.
Reports transmembrane voltage as fast, large fluorescence changes. The protein sits in the neuron's plasma membrane; when the membrane potential shifts during an action potential, a voltage-sensing domain moves and mechanically tugs a circularly-permuted GFP between bright and dim states. ASAP3 dims by ~51% per 100 mV depolarization; ASAP4e brightens by ~210% per 100 mV with much better photostability. JEDI-2P is optimized for two-photon imaging at depth.
ASAP proteins fuse two pieces: a voltage-sensing domain (VSD) from chicken (Gallus gallus) voltage-sensitive phosphatase — four transmembrane helices S1–S4 whose S4 helix carries charges that shift with membrane voltage — and a circularly permuted GFP inserted into the S3–S4 extracellular linker. In ASAP3, depolarization moves S4 outward, collapsing the cpGFP fluorophore environment and reducing fluorescence by about 51% across the physiological −70 to +30 mV range. Activation kinetics are ~2 ms at 37 °C, fast enough to track action potentials above 100 Hz in single trials. ASAP4b/4e (Lin lab, 2023) flip the response by a pair of mutations in the chromophore environment so the protein gets brighter on depolarization, and engineer the chromophore for photostability so recordings can run more than 80 seconds. JEDI-2P (St-Pierre lab, 2022) is tuned for two-photon excitation, enabling >400 µm-deep, >30-minute spike recordings in awake mice.
In plain terms
Imagine if every light bulb in your house could sense the voltage in the wires and change brightness instantly — bright when the current spikes, dim when it drops. You could map every room's electrical activity just by watching the ceiling. ASAP voltage indicators do that for neurons. Scientists give specific cells a gene that builds a protein that flashes when the cell's voltage changes. A camera then sees each neuron's electrical pulse as a tiny flash of green light.
Also called: GEVI, ASAP3 / ASAP4, JEDI-2P, fluorescent voltage sensor.
Why it matters
- Calcium indicators like GCaMP report neural activity with ~100 ms lag; GEVIs track voltage with ~1 ms resolution, revealing spike timing and synaptic integration.
- ASAP3 and ASAP4 detect subthreshold voltage swings of 10–20 mV — the dendritic computations and oscillations that calcium imaging completely misses.
- JEDI-2P enables >30-minute single-cell voltage recordings in awake, behaving mice — long enough to correlate neural codes with learned behaviors.
- Opsin-based voltage indicators heat tissue within ~12 s; ASAP4e's brighter, more photostable chromophore allows recordings 6–8× longer at lower laser power.
Where it lives
- Lin Lab, Stanford University — ASAP1 (2014), ASAP2, ASAP3 (2019), ASAP4 (2023)
- St-Pierre Lab, Baylor College of Medicine / Rice — JEDI-2P (2022), JEDI-2Psub (2025)
- Schreiter Lab, Janelia Research Campus — Ace2N-mNeon, Archon, parallel GEVI families
- Cortical neuroscience labs worldwide — ASAP3/ASAP4/JEDI-2P deployed in mice and Drosophila
Key numbers
- ASAP3 ΔF/F per 100 mV: −51% (Sub-millisecond kinetics)
- ASAP4e ΔF/F per 100 mV: +210% (Inverted polarity, high photostability)
- Activation τ_fast (37 °C): ~2–4 ms (Tracks spikes >100 Hz)
- JEDI-2P imaging depth: >400 µm (In awake mouse cortex)
- Continuous recording (JEDI-2P): >30 min (Vs. minutes for prior GEVIs)
Surprising facts
- ASAP4e's response was engineered by inverting the fluorescence-voltage relationship through just two targeted mutations in the GFP chromophore — turning a 51% dim into a 210% brighten per 100 mV.
- Older opsin-based voltage indicators required laser intensities so high (~800–1600 mW/mm²) that brain tissue heated up and recordings had to stop after ~12 seconds; ASAP4e records at ~100 mW/mm² for over 80 seconds.
- JEDI-2P captured correlated voltage signals from pairs of neurons deep in awake-mouse cortex — the first all-optical two-cell electrophysiology in a behaving animal.
- GEVIs work because the protein effectively converts a chemical-electrical signal (membrane voltage) into a photon counter — a literal voltmeter built out of a few hundred amino acids.
Open questions
- Photoactivation artifacts in ASAP4-Kv: A fraction of ASAP4e-Kv-expressing cells show anomalous fluorescence increases over time that are not driven by voltage changes — likely a light-induced protonation state change in the engineered chromophore environment. The chromophore photochemistry behind this is not fully characterized.
A short history
The first GEVI was SPARC (Bhattacharya, 2002), but slow and dim. Refinements through the 2000s improved kinetics. Lin lab at Stanford published ASAP1 in 2014 — the first GFP-VSD fusion with sub-millisecond kinetics. ASAP2s (2017) improved sensitivity. ASAP3 (2019) achieved the first in-vivo single-trial action-potential detection in awake mice. ASAP4 (2023) inverted the response, improved photostability, and enabled simultaneous one- and two-photon imaging. JEDI-2P (2022, St-Pierre lab) reached >400 µm imaging depth in awake mice.
Sources
- Lin Lab Stanford — ASAP research page (https://linlab.stanford.edu/research/recording-electricity-in-the-brain)
- Nature Methods (2023) — ASAP4 paper (https://pmc.ncbi.nlm.nih.gov/articles/PMC10627146/)
- PubMed (2022) — JEDI-2P (https://pubmed.ncbi.nlm.nih.gov/35985322/)
- bioRxiv (2021) — ASAP4 preprint (https://www.biorxiv.org/content/10.1101/2021.10.21.465345v1.full-text)
- Nature Communications (2025) — JEDI-2Psub (https://pmc.ncbi.nlm.nih.gov/articles/PMC12494759/)