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.
New methods to quantify biomolecular motility in three-dimensional cell culture models are highly demanded to advance drug development including novel immunotherapeutics. This proposal aims to develop adaptive light sheet microscopy that can image cellular interfaces in three-dimensional samples with millisecond time resolution and utilize fluorescence fluctuation spectroscopy based methods to generate maps of the paths taken by biomolecules at those interfaces. If successful, our approach will enable researchers to address many biomedical questions involving biomolecular dynamics and help accelerate drug discovery.
