Wednesday, January 15, 2025
1/15/2025
Project Summary/Abstract Two-photon laser scanning microscopy powers many projects in the BRAIN initiative because it is the best way to capture the concerted activity of living neural networks. Genetically- encoded activity probes and sensors built with fluorescent proteins are the tools of choice for labeling and recording from living circuits of neurons. However, to date, virtually all optimization of these probes has been solely in the one-photon regime. This is mainly because measuring quantitative two-photon absorption properties demands more sophisticated equipment and expertise in photophysics and nonlinear optics. The BRAIN initiative has driven many laboratories to create new genetically encoded fluorescent probes and biosensors. To characterize their multiphoton properties we created a special Resource for Multiphoton Characterization of Genetically-Encoded Probes that is supported by NIH/NINDS. This characterization is indispensable because it identifies optimum wavelengths for 2P excitation, and aids quantitative comparison of 2P brightness and 2P dynamic range (for biosensors). Over the previous funding period our Resource was used by 17 different investigators and if the grant renewed, this number will increase to 30 labs by the end of the 5th year of the next cycle. As in the previous funding period, we will provide a very broad access to our facility for Investigators whose research is relevant to the NIH/NINDS mission and to the goals of the BRAIN initiative. The information obtained will be broadly disseminated via scientific publications and two community-driven websites. We will organize annual workshops where we will provide training on 2PA photophysics and methods of measurements and provide all necessary information for researchers who were willing to start such measurements in their labs.
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