A modular dual 2-photon microscope for multimodal optogenetics, synapse biophysics and beyond

• Subject Area: Biophysics
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 413917635

By bringing molecules that respond to light, or that produce light in response to cellular activity, into the brain, scientists have begun to understand the biology of learning, sensory perception and even emotion. Encoding such molecules at the genetic level (“optogenetics”- a technique pioneered by one of the coapplicants) allows them to be expressed by specific types of cells, and in this way, a clearer picture of what nerve cells are doing emerges. But to obtain clear images from within brain tissue, or to stimulate cells at precise times and places, requires high-power illumination by infra-red pulsed lasers – a method known as two-photon microscopy. Advances in computer-controlled holography and breakthrough infrared lasers now allow the construction of a straightforward multi-channel two-photon microscope. This microscope will be able to concurrently stimulate and observe cells in 4 dimensions of space and time with sufficient precision to follow signalling in the brain. The system that we want to construct will combine a tuneable infrared laser with two finely controllable output beams, and a modular microscope for fast and targeted imaging and photostimulation of cells in brain tissue. We want to build this microscope to answer a diverse set of questions. How does the brain perceive information? What are the dynamics of connections between cells (“synapses”) and how do neurons calculate on the subcellular level? To better understand the cellular basis of cognition, we need to observe and rationally interfere with synaptic transmission and other subcellular processes. Synapses are small and operate quickly: in the millisecond regime. Their speed places extreme constraints on the optical properties of the probes used in their investigation and mandates the most sensitive illumination and detection strategies available. Therefore, we will engineer a new generation of sensitive optogenetic actuators and fluorescent reporter molecules that we will test and refine using this microscope. This microscope will create research capacity and catalyse an interdisciplinary group of scientists to engineer and use optogenetic tools with unprecedented precision and potency, to inhibit and excite cells, and to measure electrical signalling. Such tools are essential for understanding how complex behaviour, sensory perception and learning derive from cellular signalling. Finally, the microscope is designed to be a flexible platform for the ongoing development of the associated technologies. This adaptability will let us develop novel experimental themes over the course of the microscope’s lifespan by implementing new imaging modalities.

• DFG Programme: Major Instrumentation Initiatives
• Major Instrumentation: Durchstimmbares Hochleistungs-Femtosekunden-Lasersystem ; LSM-based photomanipulation system
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Humboldt-Universität zu Berlin
• Leader: Professor Dr. Andrew Plested
• Participating Persons: Professor Dr. Peter Hegemann; Professor Matthew Larkum