Multiphoton microscope

• Subject Area: Neurosciences
• Term: Funded in 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 560645340

This is an application for a replacement of a live imaging multi-photon microscope that serves the methodological core of the Hiesinger laboratory, the Neurobiology Division at FU Berlin, and the research unit RobustCircuit (currently 2022-2025) in preparation for an application for a second funding period. RobustCircuit is a consortium of 13 principal investigators with the shared goal to understand how imprecise developmental processes lead to robustness in neural circuit assembly; all projects focus on the fruit fly Drosophila melanogaster as a model system. Of the 9 complementary projects in the research unit RobustCircuit, 8 rely on long-term high-resolution live imaging of developmental dynamics in the fly brain. All developmental live imaging in the research unit is currently carried out on a multi-photon microscope in the Hiesinger lab that was purchased in 2014; the current microscope is in continuous use ~140 out of 168 hours every week, due to the nature of the long-term live imaging projects. During a planned second funding period of RobustCircuit from 2026-2029 this microscope will be 12-16 years old and neither the microscope manufacturer nor the Hiesinger group or university can ensure its continued function. The continuous availability of fly brain live imaging is paramount for the ongoing research of the groups and RobustCircuit Consortium (ideally in a second funding period, but also independent of it). Microscope time has become a limiting factor for several RobustCircuit-initiated projects in currently four research groups at the Freie University Berlin as well as collaborators within the research unit. Hence, even though we seek to replace the old microscope, we plan to continue its parallel use as long as possible during the runtime of RobustCircuit and beyond. The specific application of the instrumentation is multi-channel developmental live imaging of brain wiring at the level of individual filopodial dynamics and synapse formation in the intact Drosophila brain. This is a well-established application of multi-photon microscopy in the Hiesinger lab, which requires a minimization of phototoxicity for live imaging in two separate channels over 24 hours and longer in small regions at a depth of 50-100 micromters. Correspondingly, the microscope requirements are different from the more common application of multi-photon microscopy for functional (e.g. Ca2+) imaging in the fly brain. Such functional imaging microscopes are optimized for single channel fast imaging over shorter periods of time. By contrast, our long-term developmental live imaging requires more than one channel with separately adjustable parameter settings at higher spatial and lower temporal resolution to minimize phototoxicity over many hours. Our choice for the new instrumentation in this application is motivated by in-depth tests of three different microscopes with our own live preparations based on years-long experience with developmental live imaging.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Multiphotonen-Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Freie Universität Berlin
• Leader: Professor Dr. Peter Robin Hiesinger