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. Over the past five years, our group has informally provided this service for protein engineers and neuroscientists: they sent us newly developed proteins and we carefully characterized their 2PA properties. While this has worked in the past for just a few laboratories, the BRAIN initiative has fueled many more laboratories in need of this service to the extent that we now need support to establish a Resource. In this project, we will establish a unique Resource for Multiphoton Characterization of Genetically-Encoded Probes at the Montana State University Campus (Bozeman, MT) that will continue providing the service of characterization of 2PA properties and multiphoton stability to a much large number of protein engineers and neuroscientists. We are planning to 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 one of the microscopy 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.
Over the past few years, our group has characterized the two-photon absorption properties of genetically encoded fluorescent proteins and biosensors for a selected number of protein engineers and neuroscientists. The BRAIN Initiative has fueled many more laboratories in need of this service. In this project, we will establish a special Resource for Multiphoton Characterization of Genetically-Encoded Probes to keep our service uninterrupted and sustainable.
