# High-throughput closed-loop direct aberration sensing and correction for multiphoton imaging in live animals

> **NIH NIH K99** · CORNELL UNIVERSITY · 2024 · $104,125

## Abstract

Project Summary
 This project aims to deliver real-time aberration-corrected multiphoton imaging with improved signal-to-
noise-ratio (SNR) and spatial resolution for studying turbid deep-tissue (~2 mm) of living animals at the cellular
level. Multiphoton microscopy (MPM) has been a useful tool to study biological processes due to its high
specificity and sub-wavelength resolution. Particularly, compared to one-photon imaging, MPM uses excitation
light with a longer wavelength that penetrates deeper into tissues, while the nonlinear process requires a
multiphoton interaction that renders three-dimensional localized excitation. However, the higher-order nonlinear
excitation is more susceptible to focus aberrations, thus, posing a limit for penetration depth in highly scattering
tissues. Adaptive optics (AO) has been a promising tool for aberration sensing and correction for MPM in living
systems. However, two major issues in existing AO methods, 1) accuracy, and 2) speed in aberration sensing,
remain challenging to in vivo real-time deep-tissue imaging.
 I propose to develop a new high-throughput direct aberration sensing and correction method for MPM,
termed confocal gradient light interference microscopy (CGLIM). This technique aims to measure the aberrated
wavefront using a common-path, phase-shifting interferometer, to undo the systematic and specimen-induced
aberration which, in turn, will improve the quality of the excitation focus and enhance the signal strength.
Specifically, compared to other efforts, CGLIM uses the long-wavelength (~1.7 μm) elastic backscattered light
from tissues to directly measure the aberrated wavefront of the excitation beam, resulting in substantially lower
power compared to fluorescence techniques and eliminating photodamage, photobleaching, or heating damage
of living systems. Importantly, CGLIM measures the aberrated wavefront only near the focal plane with
nanoscale sensitivity (~2 nm or ~0.002 rad) owing to its common-path, confocal configuration. Furthermore,
CGLIM can validate the accuracy of the aberration sensing by itself via phase conjugation. Lastly, the aberration
correction procedure is directly fed by CGLIM’s measurement in a closed loop without any iterations. CGLIM is
also readily implemented in any laser-scanning system with objectives of different numeric apertures.
 With the proposed new method, I will first demonstrate aberration sensing and correction using CGLIM
with tissue phantoms and ex vivo tissue slices. Then, I will combine CGLIM with three-photon (3P) microscopy
to demonstrate aberration-corrected 3P imaging of neuronal activities in live mice and intact adult zebrafish
brains. Finally, I aim to combine the aberration-corrected MPM with the adaptive excitation source and polygon
scanning developed in-house to study real-time neuronal activities in deep regions of live brains, and T cell –
dendritic cell interactions in deep regions of a mouse’s lymph node.
 With this project, I hope to...

## Key facts

- **NIH application ID:** 10792616
- **Project number:** 5K99EB034164-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Xi Chen
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $104,125
- **Award type:** 5
- **Project period:** 2023-03-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10792616

## Citation

> US National Institutes of Health, RePORTER application 10792616, High-throughput closed-loop direct aberration sensing and correction for multiphoton imaging in live animals (5K99EB034164-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10792616. Licensed CC0.

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