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 establish my research focus on aberration-corrected interferometric multiphoton imaging for deep tissues in living animals and enable more studies in neuroscience and immunology.

Project Narrative This research will enable us to study real-time biological processes at the cellular level noninvasively in deep regions (~2 mm) of living systems. The proposed new method for aberration sensing and correction in highly scattering tissues will improve the quality of the excitation focus in multiphoton microscopy, thus, will enhance the signal strength and spatial resolution in deep-tissue imaging. In the long term, this research will allow scientists to investigate the underpinnings of brain functions and complex behaviors, as well as the mechanism of immune response in live animals.