# Head-mounted Photoacoustic Imaging of Deep-brain Neural Activities in Freely Behaving Animals

> **NIH NIH RF1** · DUKE UNIVERSITY · 2020 · $2,007,240

## Abstract

Abstract
To capture the normal brain functions, it is critically important to record the neural activities in freely-behaving
animals, with high resolution, high speed, and high throughput. So far, our knowledge about neuronal activity of
awake animals mainly relies on electrode recording, which, however, is invasive. Optical imaging techniques have
been widely used to visualize activity of a large number of neurons in mouse models using fluorescent membrane
voltage or calcium indicators. However, limited by the penetration depth (<1 mm), it is technically challenging to
record the brain functions at depths beyond the cortex layer, such as in the hippocampus. A new large-scale
recording technology with high resolution and deep penetration in freely-behaving animals would be of great utility
for the neuroscience community. Photoacoustic microscopy (PAM) is a promising candidate for this task due to
the relatively deep penetration of ultrasound waves. However, PAM has not been able to image neural activities
of freely-moving animals, because (1) it is challenging to miniaturize the imaging system, (2) there lacks calcium
or voltage probes that can report neural activities in deep brain, and (3) photoacoustic detection sensitivity of
molecular probes is traditionally low due to the strong background signals from blood. In this proposal, we plan
to overcome all of the above technical obstacles and develop head-mounted photoacoustic imaging of deep-brain
neural activities in freely-behaving animals. To achieve this goal, we will follow a three-aim strategy. (1) In Aim 1,
we will develop a miniaturized head-mounted PAM (HM-PAM) system. Several key innovations will reduce the
system footprint to 1 cm3. HM-PAM will achieve a penetration depth of ~3.0 mm with ~10−15 µm resolution, which
is deeper than that with pure optical microscopy. (2) In Aim 2, we will develop novel near-infrared photoswitchable
genetically-encoded calcium indicators (NIR-PS-GECIs) as PA probes. We will engineer and optimize a new class
of NIR-PS-GECIs based on photoacoustic Förster resonance energy transfer (FRET-PA). We have proven that
the photoswitching, which enables differential PA imaging, is currently one of the most effective approaches to
enhance the PA detection sensitivity. We will thus apply fast photoswitching of the NIR-PS-GEICs to enhance the
detection sensitivity of HM-PAM. (3) In Aim 3, the optimized HM-PAM and advanced NIR-PS-GECIs will be
thoroughly characterized and validated in dissociated neurons and in vivo. We will perform proof-of-concept
experiments of deep-brain neural activity in freely-behaving animals. In summary, our proposal will build on the
innovations of the first head-mounted PAM system, the first NIR photoswitching GECIs, and the differential FRET-
PA imaging that rejects the strong background blood signals. This enabling technology will provide a powerful
toolkit for studying neural activities in health, disease, and behavioral states.

## Key facts

- **NIH application ID:** 9924909
- **Project number:** 1RF1NS115581-01
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** Vladislav Verkhusha
- **Activity code:** RF1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,007,240
- **Award type:** 1
- **Project period:** 2020-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9924909

## Citation

> US National Institutes of Health, RePORTER application 9924909, Head-mounted Photoacoustic Imaging of Deep-brain Neural Activities in Freely Behaving Animals (1RF1NS115581-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9924909. Licensed CC0.

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