Voltage imaging (GEVI)

Optical & ultrasound (in vivo) · reads

Voltage imaging with genetically encoded indicators is fluorescence microscopy of neurons that carry a protein sensor whose brightness changes with membrane voltage; two-photon excitation reaches the sensor in deep tissue. What it reads is voltage-dependent fluorescence, an indirect measure from which spikes and slower subthreshold changes are inferred. In mice, viral delivery confined the indicator to excitatory neurons of visual cortex layer 2/3, enriched at their cell bodies and nearest dendrites, and traces from awake animals yielded inferred spikes and slow voltage changes; in larval zebrafish, spike-like transients were detected.

Spatial resolution

40–60 µm (two-source)

Temporal resolution

8 ms (recoverable timing)

Field of view

18685000 µm³ (acquired volume)

Neurons covered

180 neurons (sensing envelope)

Reads

voltage-dependent fluorescence; inferred action potentials; inferred subthreshold membrane-voltage fluctuations

Writes

n/a

Destructive?

minimally destructive

Prerequisites

germline genetic engineering: Mouse inherited-Cre protocol: use Slc17a7-Cre mice.; genetic engineering: Mouse inherited-Cre protocol: inject Cre-dependent JEDI-2P AAV into visual cortex.; anesthesia: Mouse inherited-Cre protocol: use isoflurane surgical anesthesia with analgesia and local bupivacaine.; surgery: Mouse inherited-Cre protocol: open a craniotomy over the injection site, seal it with a coverslip, and cement a washer headbar to the skull.; other: Mouse inherited-Cre protocol: immobilize the head through the attached headbar.; genetic engineering: Mouse viral-Cre protocol: co-inject Cre AAV and Cre-dependent JEDI-2P AAV into visual cortex of wild-type C57BL/6J mice.; anesthesia: Mouse viral-Cre protocol: use Zolethil-Xylazine surgical anesthesia with preoperative buprenorphine.; surgery: Mouse viral-Cre protocol: place and cement a coverslip over the targeted cortex and cement an aluminum head plate to the skull.; other: Mouse viral-Cre protocol: habituate mice progressively to head fixation before recording.; germline genetic engineering: Larval zebrafish protocol: use fluorescence-positive F2 transgenic reporter larvae on a casper or nacre background at 4–6 days post-fertilization.; other: Larval zebrafish protocol: paralyze larvae with pancuronium bromide and confirm paralysis by absent movement after tactile stimulation.; other: Larval zebrafish protocol: expose paralyzed larvae to PTZ, embed them in low-melting-point agarose, and maintain PTZ as the immersion medium during imaging.

Theoretical spatial resolution

370 nm (point spread)

Theoretical field of view

127200000 µm³ (reachable envelope)

Description

Genetically encoded voltage indicators are protein biosensors whose fluorescence brightness changes with membrane voltage. Indicators built by coupling a fluorescent protein to a voltage-sensitive phosphatase domain combine voltage sensing and fluorescence modulation in one genetically encoded molecule. Their fluorescence-versus-voltage relation can be sigmoidal, consistent with voltage-sensitive-domain behavior. Two-photon optical excitation permits visualization in deep tissue, while fluorescence detection measures voltage-dependent brightness changes that report neuronal voltage dynamics. In a mouse protocol, Cre-dependent AAVs delivered indicator genes to visual-cortex layer 2/3 in transgenic mice expressing Cre in excitatory cells. A short Kv2.1-derived peptide restricted the indicator to somata and proximal dendrites to minimize neuropil background. Ring-like fluorescence was consistent with plasma-membrane localization and somatic enrichment. This genetic targeting confines expression to the Cre-defined cell population, and two-photon visualization provides optical access to fluorescence from labeled neurons in tissue. Within this scope, detected fluorescence brightness tracks neuronal membrane voltage, while the expression strategy determines cell-type targeting and subcellular localization.

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