# Multi-layer neuronal imaging with reverberation multiphoton microscopy

> **NIH NIH R01** · BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) · 2022 · $400,516

## Abstract

ABSTRACT
 Many brain areas, such as neocortex and olfactory bulb, are vertically organized into
layers containing distinct cell types that show different activity profiles and project to
different downstream targets. Fast, volumetric imaging is thus indispensable to capture
the dynamics of such neuronal populations within their stratified environments. While
multiphoton microscopy (MPM) has become the gold standard for high resolution
imaging from deep within brain tissue, it is generally restricted to 2D planar imaging. We
propose to develop a technique to perform volumetric MPM where a long-range z-stack
is acquired by near-instantaneous axial scanning, while maintaining 3D micron-scale
resolution. Our technique, called reverberation MPM, enables the monitoring of
neuronal populations over large scales, including the depth scale, with no speed penalty
compared to conventional MPM.
 Reverberation MPM is a new technique which we have demonstrated only recently
with proof of principle two-photon experiments. Much of our proposal will be focused on
further developing this tool and characterizing its performance. Moreover, we propose to
extend our technique to three-photon microscopy, for increased depth penetration. Our
goal is to perform comprehensive 3D-resolved imaging of neuronal populations within
volumes up to 1×1×1mm3, spanning the entire thickness of the mouse cortex.
 A key advantage of reverberation MPM is its extreme simplicity. It requires only the
addition of a reverberation loop to a conventional MPM equipped with fast detection
electronics. Moreover, it allows the acquisition of an arbitrary number of planes without
increasing setup complexity. Other advantages are that our system is light efficient and
easily compatible with video-rate scanning, making it ideal for volumetric calcium
imaging using genetically encoded calcium indicators. These advantages make
reverberation MPM particularly attractive as a general tool for fast, high resolution,
large-scale volumetric imaging in brain tissue.

## Key facts

- **NIH application ID:** 10320482
- **Project number:** 5R01NS116139-03
- **Recipient organization:** BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
- **Principal Investigator:** Jerome Mertz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $400,516
- **Award type:** 5
- **Project period:** 2020-03-15 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10320482

## Citation

> US National Institutes of Health, RePORTER application 10320482, Multi-layer neuronal imaging with reverberation multiphoton microscopy (5R01NS116139-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10320482. Licensed CC0.

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