# Fluorescence Fluctuation Spectroscopy with Light Sheet Microscopy

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA-IRVINE · 2021 · $196,250

## Abstract

PROJECT SUMMARY
Three-dimensional (3D) cell culture models have been demonstrated to behave more similar to animal models
than flat cell monolayers. The high physiological relevance of those human-on-a-chip type assays is key to
accelerate drug development. To efficiently image 3D specimen, slow single point laser scanning microscopy
is being replaced by camera-based light sheet microscopy for its superior imaging speed and reduced light
exposure. While light sheet microscopy has been successfully applied to image dynamic processes on larger
scales such as cell migration in Drosophila, zebrafish, and C. elegans embryos, resolving dynamics on the
molecular level has been mostly neglected. However, measuring biomolecular dynamics is very important to
understand cell signaling, cellular responses, spatial organization of cell surface receptors, and, especially,
how cells engage in interactions with surrounding cells – mechanisms that can be targets of new drugs
including immunotherapeutics. Hence, we propose to develop novel approaches to quantify molecule/particle
movement in three-dimensional cell culture models and tissues with light sheet imaging. This high risk/high
reward proposal will tailor light sheet imaging to study cellular interfaces with high spatiotemporal resolution
and leverage two-dimensional pair correlation analysis to map the paths of biomolecules taken at those
interfaces.
In aim 1, we will use fast beam scanning, steering, and refocusing to generate tipped/tilted and curved light
sheets tailored to cellular interfaces. Imaging of one or a few complex planes using micromirror-based adaptive
optics in the detection path will allow us to record data at a much higher rate than possible with conventional z
stacks comprised of many planes. Overall feasibility is indicated by previous use of beam scanning, steering
and refocusing to track single particles on the millisecond timescale with the orbital tracking approach.
In aim 2, we will study the spatial organization of molecule dynamics in the presence of barriers or obstacles at
cellular interfaces. To reveal those barriers with single pixel resolution, we recently suggested the two-
dimensional pair correlation function (2D-pCF) approach but, so far, a successful application of this method to
3D cell culture models is lacking. Hence, we intend to prove the effectiveness of this new strategy with light
sheet microscopy in the more challenging case of cell-cell contacts/interactions. In many biomedical studies
involving cell-cell contacts, membrane receptors are the focus of interest. Therefore, we will utilize a model of
natural killer cells interacting with target cancer cells to develop our approach.
Our goal is to enable researchers to efficiently study cellular interactions in 3D specimen on the molecular
level, which are especially important for the development of innovative immunotherapy approaches, for
example, to treat cancers.

## Key facts

- **NIH application ID:** 10242938
- **Project number:** 5R21GM135493-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** ENRICO GRATTON
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $196,250
- **Award type:** 5
- **Project period:** 2020-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10242938

## Citation

> US National Institutes of Health, RePORTER application 10242938, Fluorescence Fluctuation Spectroscopy with Light Sheet Microscopy (5R21GM135493-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10242938. Licensed CC0.

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