2-Photon microscope (in-vivo)

• Subject Area: Neurosciences
• Term: Funded in 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 502050812

Visualizing neuronal activity in the living brain is crucial for understanding how neuronal circuits guide behavioural decisions. At the Biology department of the Freie Universität Berlin (divisions of Neurobiology and Neurogenetics), we are planning to acquire a 2-photon microscope optimized for the functional characterization of neuronal cell types and their synaptic connections in the brain of living fruit flies (Drosophila melanogaster). The contributing research groups already use similar molecular genetic techniques for labelling and manipulating specific neuron types involved in different tasks ranging from vision, navigation, locomotion, all the way to the ageing, learning and memory. Our common goal is to understand how specific cell types respond to clearly defined stimuli (like different visual or olfactory cues), and how these signals are then transformed via synaptic transmission towards the central brain. We will visualize neuronal activity using existing genetically encoded indicators of activity not only in wild type specimens, but also after introducing different molecular and environmental manipulations affecting the development and/or function of neurons in a controlled way. Importantly, all research groups share a common focus on understanding how variability in neuronal properties shape the robustness of behavioural outcomes. For the planned use, the two-photon microscope must provide a sufficiently large working space underneath the objective. This excludes those ‘off the shelf’ multiphoton laser scanning systems where motorized stage and condenser block most of the desired space. A window will be cut into the head capsule of living flies (using a stereo microscope placed in close vicinity to the microscope, as a ‘mounting station’). Flies will then be placed under the microscope’s objective in a way such that the animal can experience different stimuli (visual or olfactory). These experiments will require the fly to be able to flap its wings while maintaining its normal flight posture (virtual flight), or to be placed on a spherical treadmill. Hence, the planned instrumentation must provide the necessary space for implementing this. Furthermore, several of the proposed experiments require visual stimulation, either using stationary LEDs (simulation of polarized skylight), or rotating LEDs (simulation of celestial bodies), or an LED matrix (simulation of different landmarks), or several of these in combination. These stimuli are rather bulky and require a large working space under the objective. The whole working space must be optically sealed (due to the use of UV light for stimulation) and must be protected against vibrations. We are planning for two GaAsP detectors for optimal performance. A tunable laser (680 nm – 1080 nm) will be used for combining activity imaging (for instance GCaMP-type Calcium imaging) and activating neurons using red-shifted Channelrhodopsin (CsChrimson, for instance).

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: 2-Photonmikroskop (in vivo)
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Freie Universität Berlin
• Leader: Professor Dr. Mathias Wernet