A red-emitting, genetically encoded indicator for two-photon voltage recording in vivo

This paper introduces VADER1, a novel red-emitting, genetically encoded voltage indicator that overcomes previous limitations to enable reliable, deep-tissue, two-photon recording of neuronal action potentials in vivo while supporting spectral multiplexing with other sensors and optogenetic tools.

The Gist

Imagine the brain as a bustling city where billions of tiny messengers (neurons) communicate by sending rapid electrical sparks. To understand how this city works, scientists need a way to "listen" to these sparks without breaking the walls of the city.

For a long time, scientists had a special tool called a GEVI (Genetically Encoded Voltage Indicator). Think of this as a tiny, invisible security camera that scientists can program into specific types of neurons. When a neuron fires an electrical spark, this camera flashes, allowing scientists to see the activity in real-time.

However, there was a major problem with the existing cameras:

• The Color Problem: Most of these cameras flashed in blue or green light. But to see deep inside the brain (like looking into the basement of a skyscraper), scientists need to use a special "flashlight" called two-photon excitation. Blue and green light get blocked by the brain's tissue, like trying to see through a thick fog with a weak flashlight.
• The Red Gap: Scientists needed a camera that glowed red. Red light is like a powerful laser that can cut through the fog and reach deep into the brain. Plus, red light plays nicely with other tools (like blue-light switches used to control neurons), allowing scientists to use multiple tools at once without them interfering.

The Problem: Until now, the red cameras that existed were too dim or slow to work well with this deep-tissue "two-photon" flashlight. They were like trying to take a clear photo of a fast-moving car in the dark with a broken camera; the results were blurry or non-existent.

The Solution: VADER1
This paper introduces a new invention called VADER1. Think of VADER1 as a brand-new, high-performance red security camera designed specifically for deep-tissue work.

Here is what VADER1 can do that the old ones couldn't:

• Deep Vision: It works perfectly with the two-photon flashlight, allowing scientists to see electrical sparks clearly even in the deepest layers of the brain (specifically mentioned: Layer 5 of the cortex).
• Fast & Flexible: It is fast enough to catch the split-second sparks of a neuron firing. It works whether the scientists are scanning the brain randomly (like checking specific houses) or scanning it quickly in a sweeping motion (like a security guard doing a full patrol).
• Team Player: Because it glows red, it doesn't clash with other tools. Scientists can use VADER1 to watch electrical sparks while simultaneously using a different tool to watch calcium levels (another sign of brain activity), creating a "dual-color" view of what's happening.

The Bottom Line
VADER1 fills a critical missing piece in the puzzle. It finally allows scientists to combine deep-brain imaging with the ability to watch fast electrical signals and other neural events at the same time. This sets the stage for a new era of "all-optical electrophysiology," where scientists can both watch and control the brain's electrical city using only light, without needing to insert physical wires.

Technical Summary

Technical Summary: VADER1 for Two-Photon Voltage Imaging In Vivo

Problem Statement
Genetically encoded voltage indicators (GEVIs) offer a powerful means to measure neuronal membrane potential with cell-type specificity and millisecond temporal resolution in a minimally invasive manner. While red-shifted GEVIs are theoretically ideal for in vivo applications—specifically because they allow for spectral multiplexing with other sensors and compatibility with blue-light-activated opsins for all-optical circuit interrogation—their practical utility has been severely limited. Existing red GEVIs demonstrate poor performance under two-photon (2P) excitation, the standard modality for deep-tissue imaging. Consequently, there has been a critical gap in the ability to perform reliable, deep-tissue voltage recordings using red-shifted indicators.

Methodology and Development
To address this limitation, the authors developed VADER1, a new red-shifted GEVI specifically engineered for high performance under two-photon excitation. The study evaluates VADER1's capabilities using both random-access and resonant-scanning microscopy modalities. The researchers tested the indicator's efficacy in in vivo settings, focusing on its ability to detect action potentials (spikes) and its performance at varying cortical depths. Furthermore, the study investigated the feasibility of dual-color imaging by combining VADER1 with calcium indicators.

Key Results
The introduction of VADER1 successfully overcomes the performance barriers of previous red GEVIs under 2P illumination. The key findings include:

• Reliable Spike Detection: VADER1 enables the reliable detection of neuronal spikes in living tissue under two-photon excitation.
• Deep-Tissue Imaging: The indicator supports voltage recordings from neurons located as deep as cortical layer 5.
• Microscopy Compatibility: VADER1 functions effectively across different imaging modalities, including both random-access and resonant-scanning microscopy, allowing for extended voltage imaging sessions.
• Spectral Multiplexing: The indicator permits dual-color imaging when paired with calcium indicators, demonstrating its utility in multi-parameter experiments.

Significance and Claims
The paper positions VADER1 as a critical tool for filling a specific spectral gap in neuroimaging. By enabling integrated optical measurements of fast electrical activity alongside other neural signals (such as calcium dynamics) in deep brain regions, VADER1 establishes a foundation for two-photon all-optical electrophysiology. The authors claim that this advancement facilitates more comprehensive, minimally invasive, and cell-type-specific interrogation of neural circuits in vivo, overcoming the historical performance limitations of red-shifted indicators in two-photon microscopy.