# Simultaneous two-photon imaging and two-photon manipulation of neural activity in freely behaving mice

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2024 · $187,831

## Abstract

Simultaneous two-photon imaging and two-photon manipulation of neural activity in freely-
behaving mice
The ability to record and manipulate neural activity in freely-behaving mice is a key step to understand the
neural circuits under natural behavior. One-photon miniscope has become a mature technology and it can
perform calcium imaging and optogenetics simultaneously while the mice freely behave. Though it has
enabled many new discoveries in neuroscience, the ability to image and manipulate neural activity deeper
into the tissue and in higher spatial specificity can further advance the field and enable studying many new
questions. Compared to one-photon, two-photon techniques can access much deeper tissue and have a
much higher spatial specificity. However, it has been challenging to integrate high performance optics into
a compact footprint miniscope to perform simultaneous two-photon imaging and two-photon optogenetics,
due to the excessive challenges in optical and mechanical design.
Here, we propose a new miniscope that can simultaneously perform two-photon calcium imaging and two-
photon optogenetics in freely-behaving mice. We innovatively integrate two different beam forming
techniques in the miniscope for the imaging beam and optogenetics beam. This unique combination
enables a very compact mechanical design and a high optical performance. Crucially, we will achieve a
high-spatiotemporal-resolution in imaging, and patterned stimulation in optogenetics where a group of
user-selected neurons could be simultaneously photostimulated. This allows us to manipulate the
ensemble activity while monitoring the response of the neural circuit, all in cellular resolution. The entire
device can have a dimension <~14x14x25 mm3 and a weight <3 g, suitable to be mounted on the skull of
freely-moving mice. Furthermore, we will custom design and manufacture the optics to support a large field
of view of 400 µm in diameter. This allows us to access a large amount of neurons. Finally, the focal depth
of both beams could be controlled independently so we could image and manipulate the neural activity
across a 3D brain volume.
The success of this project will create a two-photon miniscope that can simultaneously image and
manipulate neural activity, both in cellular resolution, high temporal resolution/specificity and over a large
3D volume deep in the brain tissue, in freely-behaving mice. The proposed miniscope could enable new
research that is previously not possible, such as investigating the neural circuits of 2D navigation and
social behavior. Our miniscope will greatly benefit the neuroscience community, and be readily deployed
to many research labs. While we will design and test the miniscope for mice, its application could be
extended to rats and non-human primates in future.

## Key facts

- **NIH application ID:** 10993452
- **Project number:** 1R21EB035306-01A1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Weijian Yang
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $187,831
- **Award type:** 1
- **Project period:** 2024-08-15 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10993452

## Citation

> US National Institutes of Health, RePORTER application 10993452, Simultaneous two-photon imaging and two-photon manipulation of neural activity in freely behaving mice (1R21EB035306-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10993452. Licensed CC0.

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