# Designing and deploying an expanded color palette of voltage indicators engineered for multiphoton microscopy

> **NIH NIH U01** · BAYLOR COLLEGE OF MEDICINE · 2020 · $112,013

## Abstract

PROJECT SUMMARY/ABSTRACT
The ability to track electrical activity in genetically defined neurons deep in the brain has long been sought by neuroscientists
to unravel the functions of neuronal circuits in health and disease. We seek to address this technical gap by developing
genetically encoded voltage indicators (GEVIs), which are fluorescent proteins that report voltage dynamics as changes in
brightness. GEVIs would be excellent completements to broadly-used indicators of calcium, another important information
carrier in the brain. For example, GEVIs promise to report forms of neural activity that are not generally reported by calcium
indicators in typical mammalian cortical neurons: hyperpolarizations, subthreshold depolarizations, and fast trains of action
potentials. While these genetically encoded voltage indicators have shown early promise for detecting voltage dynamics,
improvements in their brightness, photostability, and responsivity to fast voltage changes are needed for robust imaging in
mammalian systems. Moreover, current GEVIs have particularly low performance under two-photon microscopy, the
method of choice for deep-tissue imaging.
 The overall goal of this proposal is to develop a color palette of voltage indicators optimized for two-photon
microscopy. We first propose to develop a new microscopy platform that can rapidly screen indicator variants by
automatically monitoring and analyzing their fluorescence responses to fast voltage transients. Second, we propose to
develop activity-specific GEVIs, that is, indicators that are optimized for specific forms of neural activity. Specifically, we
will focus on optimizing a GEVI for detecting spikes and a GEVI for monitoring synaptic activity, that is, graded potentials
about the resting membrane potential. Finally, we propose to expand the color palette of voltage indicators to enable two-
photon multi-color imaging and all-optical electrophysiology. Voltage indicators with improved performance will be
comprehensively characterized across all key metrics in acute brain slices and in vivo to facilitate deployment in downstream
applications. We anticipate that this research project will produce voltage indicators with robust performance and with broad
applicability in neuroscience, enabling voltage imaging of neuronal ensembles with cell type specificity and high
spatiotemporal resolution.

## Key facts

- **NIH application ID:** 10169033
- **Project number:** 3U01NS113294-02S1
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Francois St-Pierre
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $112,013
- **Award type:** 3
- **Project period:** 2019-08-15 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10169033

## Citation

> US National Institutes of Health, RePORTER application 10169033, Designing and deploying an expanded color palette of voltage indicators engineered for multiphoton microscopy (3U01NS113294-02S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10169033. Licensed CC0.

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