Friday, April 4, 2025
4/4/2025
Fast multicolor confocal fluorescence imager for neural network mapping - Project Summary/Abstract Brain functions are driven by highly dynamic neuronal activities operating within a complex neural network. Impairment of neuronal connections during development causes neurodevelopmental disorders such as attention-deficit/hyperactive disorder (ADHD) and autism spectrum disorder (ASD), which affect learning ability and social skills. To identify the normal/abnormal connections of neurons and track individual neurons at the sub-cellular levels and millisecond time scales for the investigation of the mechanisms of neurodevelopmental disorder, multicolor barcode methods have been developed to label adjacent neurons with different fluorophores. In addition, fluorescence imaging techniques that acquire multicolor spatial information have emerged. However, there is no tool that enables simultaneous multicolor imaging at high temporal resolution (>140 Hz) to capture the fast dynamics occurring between neurons and possible cellular deformations or conformational changes in live, moving animals. Moreover, high spatial resolution and optical sectioning capabilities are necessary since neurons are concentrated in an optically dense 3-D environment and detailed sub-neuronal structures need to be resolved to observe neuronal connectivity. During this R&D program, Physical Sciences Inc. (PSI), in collaboration with UMass Chan Medical School (UMCMS), proposes to develop and demonstrate a high-speed (>140 Hz frame rate) multicolor confocal fluorescence imager that can simultaneously capture multi-wavelength signals and display them as separated or merged multicolor images and 3-D stacks. The proposed imager will be used to map complex neuronal network activity and identify individual neurons in moving organisms. Technology innovations include a novel spatiotemporally modulated (STM) imaging technology, which enables both confocal scanning and simultaneous multi-wavelength excitations. The Phase I effort will focus on 1) developing a fast multicolor confocal prototype imager employing four laser excitations; 2) validating the system with four different fluorescent beads to demonstrate its capabilities of simultaneous multicolor imaging and optical sectioning at high temporal resolution; 3) performing in vivo experiments with “NeuroPAL” (a Neuronal Polychromatic Atlas of Landmarks) C. elegans wherein neurons express four (or more) different fluorescent proteins that result in multicolor labeling based on stereotyped combination of neuron-specific reporters that drive the expression of different colored fluorophores. These studies will demonstrate the technology feasibility, and demonstrate its value to support a broad spectrum of neuroscience research efforts. This R&D project will lay the foundation for the development and commercialization of a multicolor imaging technology with high spatiotemporal resolution to study and understand of real-time neuronal dynamics in live animals, offering a critical tool for neuroscience research.
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