A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo

The paper introduces FORCE1s, a versatile, positive-going genetically encoded voltage indicator engineered to enable robust, spike-resolved two-photon imaging across diverse platforms, including standard microscopes and affordable miniscopes, thereby democratizing deep-tissue voltage recording in vivo.

The Gist

Imagine trying to listen to a single person whispering in a crowded, noisy stadium. That is essentially what neuroscientists have been trying to do for years: listen to the tiny electrical whispers (action potentials) of individual brain cells while they are deep inside a living, moving animal.

For a long time, this was nearly impossible. The tools they had were either too slow, too dim, or required expensive, specialized equipment that only a few labs in the world possessed.

This paper introduces a new tool called FORCE1s. Think of it as a revolutionary new "microphone" for the brain that finally makes listening to these electrical whispers easy, loud, and accessible to everyone.

Here is the breakdown of how it works, using simple analogies:

1. The Problem: The "Dim Lightbulb" and the "Fast Blink"

Before FORCE1s, scientists used sensors that worked like dim lightbulbs that flickered off when a neuron fired.

• The Dimness: When a neuron fired, the light got slightly dimmer. In a noisy room (the brain), it's hard to notice a lightbulb getting a little darker.
• The Speed: These sensors turned off so fast (in a fraction of a millisecond) that standard cameras couldn't keep up. It was like trying to take a photo of a hummingbird's wings with a slow shutter speed; you just get a blur.
• The Result: You needed incredibly expensive, super-fast cameras (microscopes) to catch the signal, and even then, you could only watch one or two neurons at a time.

2. The Solution: The "Bright Flashlight"

The researchers engineered FORCE1s to solve these two problems.

• Positive-Going (The Flashlight): Instead of getting dimmer, FORCE1s gets brighter when a neuron fires. Imagine a neuron holding a flashlight. When it fires, it suddenly blasts a bright beam of light. It is much easier to spot a sudden bright flash against a dark background than a slight dimming.
• The "Goldilocks" Speed: The old sensors were too fast for standard cameras. FORCE1s was engineered to be fast enough to catch the spike, but just slow enough that a standard, affordable camera can still see it clearly. It's like tuning a radio to a frequency where the signal is strong and clear, rather than static.

3. The Results: From One Ear to a Whole Orchestra

Because FORCE1s is so bright and "friendly" to standard cameras, the results are game-changing:

• Standard Microscopes: Labs that already have standard microscopes (the kind found in most biology departments) can now do deep-tissue voltage imaging. They don't need to buy a $2 million machine.
• The "Group Shot": Previously, scientists could only watch one neuron at a time. With FORCE1s, they can now watch dozens of neurons at once, like watching a whole orchestra play instead of just one violinist.
• Freely Moving Animals: The team even put this sensor into a tiny, backpack-sized microscope (a "miniscope") that mice can wear. For the first time, they could record the electrical activity of neurons while the mice were running around, exploring, and behaving naturally. It's like finally being able to record a conversation while the people are walking down the street, rather than forcing them to sit still in a soundproof booth.

4. Why This Matters

Think of the brain as a massive, complex city.

• Before: We could only see the city from a helicopter, and only when the weather was perfect, and only for a few minutes at a time.
• With FORCE1s: We can now walk the streets, listen to the conversations in real-time, watch how the traffic flows, and see how the city changes when the citizens are going about their daily lives.

In short: The authors have built a better, brighter, and more versatile "flashlight" for the brain. This tool democratizes neuroscience, allowing researchers everywhere to finally see the electrical language of the brain in action, in real-time, and in living, moving animals.

Technical Summary

1. Problem Statement

Genetically encoded voltage indicators (GEVIs) are essential for optically measuring membrane potential dynamics with cell-type specificity. However, two-photon (2P) voltage imaging has remained inaccessible to most neuroscience laboratories due to two primary barriers:

• Ultrafast Kinetics vs. Sampling Speed: Existing high-performance GEVIs (e.g., JEDI-2P) exhibit ultrafast repolarization kinetics. To detect action potentials (spikes) with high fidelity, these indicators require sampling rates near or above 1 kHz. Most standard resonant-scanning microscopes cannot achieve these speeds over fields of view (FOV) large enough for multi-cell recordings.
• Low Signal-to-Noise Ratio (SNR): Current indicators often have small fluorescence responses ($\Delta F/F$). High sampling rates reduce photon counts per timepoint, increasing noise. Furthermore, "bright-to-dim" indicators (which darken upon depolarization) suffer from high baseline photon shot noise from the neuron and surrounding neuropil, further degrading spike detectability.

These limitations restrict deep-tissue, multi-cell voltage imaging to specialized, expensive microscopy platforms (e.g., FACED, ULoVE), preventing broad adoption.

2. Methodology

The authors developed FORCE1s (Fluorescent Observer of Rapidly Climbing Electropotentials 1), a new GEVI designed to overcome these kinetic and SNR barriers.

• Design Strategy:

  • Parent Sensor: Started with JEDI-2P, a proven "bright-to-dim" indicator.
  • Polarity Inversion: Through saturation mutagenesis of the voltage-sensing domain (VSD) and the GFP chromophore, the authors inverted the response polarity. They identified the S151D mutation (adjacent to the chromophore-interacting residue E152) as the key switch, converting the sensor to a "dim-to-bright" (positive-going) indicator that fluoresces more intensely during depolarization.
  • Optimization: The final variant, FORCE1s, contains eight mutations relative to JEDI-2P. It was engineered to balance response amplitude, brightness, photostability, and kinetics.
  • Targeting: A Kv2.1-derived motif was added (FORCE1s-Kv) to ensure plasma membrane localization and somatic enrichment.

• Screening & Characterization:

  • In Vitro: High-throughput 2P screening in HEK293 cells using electric field stimulation. Characterized excitation spectra, brightness, photostability, and kinetics (on/off rates) via whole-cell patch-clamp and 1P/2P imaging.
  • In Vivo: Validated in mouse visual cortex (V1) using:
    • Resonant-scanning microscopes (standard 400–440 Hz).
    • ULoVE (Ultrafast Local Volume Excitation) for single-cell, ultrafast (7.1 kHz) recordings.
    • Diagonal Scanning (AOD-based) for multi-cell, sub-kHz (200–250 Hz) imaging.
    • FACED 2.0 for large-scale (429 × 532 µm) imaging at 256 Hz.
    • MINI2P (Miniscope) for freely moving mice.

3. Key Contributions

• FORCE1s Indicator: A green, positive-going GEVI that brightens upon depolarization.
• Kinetic Tuning: Unlike previous indicators that require >1 kHz sampling, FORCE1s has repolarization kinetics (~2.9 ms) that are slow enough to allow reliable spike detection at sub-kilohertz frame rates (200–400 Hz) but fast enough to resolve individual spikes.
• Democratization of 2P Voltage Imaging: Demonstrated that robust, spike-resolved multi-cell recordings are possible on standard resonant-scanning microscopes, removing the need for specialized ultrafast hardware for many applications.
• Versatility: Validated across multiple modalities, from standard resonant scanners to advanced FACED and compact MINI2P miniscopes.

4. Key Results

• Superior Performance In Vitro:

  • Response Amplitude: FORCE1s showed a ~100% $\Delta F/F$ response to spikes, significantly outperforming JEDI-2P (26%) and ASAP4e (34%).
  • Kinetics: Exhibited a dominant 2.8 ms on-kinetic component and a 2.4/9.1 ms off-kinetic profile. This "slower" off-kinetic allows photon accumulation, enhancing SNR at lower frame rates.
  • Brightness & Stability: >2x brighter than ASAP4e and retained 39% of JEDI-2P's baseline brightness (despite being dim-to-bright). Showed improved photostability.

• In Vivo Validation (Anesthetized & Awake):

  • Resonant Scanning (440 Hz): FORCE1s-Kv detected spikes with high fidelity (F1 score of 0.87) compared to JEDI-2P-Kv (0.48). It successfully tracked subthreshold dynamics and burst firing.
  • ULoVE (7.1 kHz): FORCE1s-Kv produced 3.4-fold larger spike amplitudes than JEDI-2P-Kv. It enabled reliable detection of bursts (up to 326% response) and subthreshold fluctuations (1.8x larger).
  • Multi-Cell Imaging:
    • Diagonal Scanning: Enabled 1-hour recordings of 8–10 neurons simultaneously at 210–250 Hz in awake mice.
    • FACED 2.0: Expanded the FOV by ~3.6x (imaging 154 neurons) at 256 Hz while maintaining spike fidelity, a feat impossible with JEDI-2P at equivalent speeds due to spike loss.
    • Standard Resonant: Simultaneously imaged 6 neurons at 400 Hz with high SNR.

• Freely Moving Imaging (MINI2P):

  • Successfully recorded single-spike resolution voltage dynamics in freely moving mice for 40+ minutes using a compact, commercially available miniscope.
  • Maintained high SNR (~6.3) and photostability (retaining ~72% fluorescence) throughout sessions.
  • Demonstrated functional relevance by mapping border-tuning (spatial firing fields near arena walls).

• Multiplexing:

  • Successfully co-imaged FORCE1s-Kv (voltage) and a red-shifted acetylcholine sensor (rACh1h), revealing correlations between low-frequency voltage oscillations and cholinergic brain states.

5. Significance

This work represents a paradigm shift in optical voltage imaging:

1. Accessibility: By tuning the kinetics to match the capabilities of standard resonant-scanning microscopes, FORCE1s makes deep-tissue, multi-cell voltage imaging accessible to the broader neuroscience community, not just those with specialized ultrafast setups.
2. Scalability: It enables the recording of larger neuronal populations (dozens to hundreds) and larger fields of view without sacrificing spike detection accuracy.
3. Behavioral Relevance: The compatibility with MINI2P miniscopes allows for the study of voltage dynamics during naturalistic, freely moving behaviors, bridging the gap between cellular resolution and ethologically relevant contexts.
4. Toolbox Expansion: FORCE1s serves as a versatile scaffold for future engineering, offering a "community-ready" tool that democratizes the mapping of neuronal computation across diverse experimental contexts.