# Direct wavefront sensing and adaptive optics to enable two-photon imaging axons and spines throughout all of cortex

> **NIH NIH U24** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2022 · $340,052

## Abstract

Principal Investigator (Last, first, middle):KLEINFELD, DAVID
Project summary
 Two-photon laser scanning microscopy is indispensable for imaging the structure and function of the
mammalian brain with subcellular resolution. However, the resolution and efficiency decreases with tissue
depth as a result of scattering and optical aberrations. Adaptive optics can improve multi-photon imaging by
synthesizing a distortion to the wavefront of the excitatory beam that compensates for aberrations in the
wavefront that are created by the tissue. Our system utilized adaptive optics to enable investigators to probe
subcellular dynamics in individual synapses along the full depth of cortex. This is a crucial advance, particularly
as layer 5 and 6 cortical output neurons lie deep to the surface of the brain. Many contemporary studies within
the neuroimaging community are limited by the current inability to record synaptic dynamics within output
regions of cortex, e.g., layer 5, as opposed to within the dominant input layer, i.e., layer 4, and the intermediate
levels, e.g., layers 2/3. We will remove this limit and thus open up a new subfield of in vivo studies on
subcellular determinates of cortical output.
 Our proposed work incorporates "good engineering practice" in the design of our current adaptive optics
two-photon microscope design. We will disseminate accurate plans and construction details to enable other
laboratories to duplicate this system. We will further educate the neuroimaging community on the principles of
adaptive optics and the design and utility of adaptive optics-based two-photon microscopes. This effort includes
workshops at UC San Diego. Throughout the period of the proposed grant, we will continue to advance the
adaptive optics two-photon system and update and expand our user base. Proposed new directions include a
rapid shift in focus together with aberration correction for diffraction limited focus over planes separated by as
much as 300 µm and the incorporation of a resonant scanner for fast cell-based imaging. Lastly, we will form a
team effort among users and incorporate feedback from the team to extend adaptive optics into new areas of
inquiry in neuroscience as they arise.

## Key facts

- **NIH application ID:** 10425220
- **Project number:** 5U24EB028942-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** David Kleinfeld
- **Activity code:** U24 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $340,052
- **Award type:** 5
- **Project period:** 2019-09-30 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10425220

## Citation

> US National Institutes of Health, RePORTER application 10425220, Direct wavefront sensing and adaptive optics to enable two-photon imaging axons and spines throughout all of cortex (5U24EB028942-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10425220. Licensed CC0.

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