Project Details
Projekt
2-photon microscope
Subject Area
Neurosciences
Term
Funded in 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 546794481
Animals interact every day with a complex environment, in which sensory cues are often overlapping in variable combinations. However, animals often display striking capabilities to distinguish relevant changes in the sensory environment, even when they are small in relation to the total sensory information at a particular time. In the brain, sensory information is represented by firing patterns of defined neuronal populations. We are utilizing the comparatively small brain of the common fruit fly Drosophila to address how olfactory information is represented in higher brain centres. Concentrating on an exemplary neuronal circuit that shares many organizational and functional characteristics with centres in the brain of vertebrates, including mammals- we investigate how codes of neuronal activation that represent olfactory stimuli are formed and how they can be modified. The patterns of neuronal activation in response to olfactory stimuli can only be recorded in the live animal exposed to various stimuli. In addition, their changes in dependence of stimuli combination, modified stimulus value and precedent experience- can only be recorded in a behaving animal. The setup we request will be essential for these experiments. Two-photon microscopy (2P-microscopy) allows the recording of dynamic changes in fluorescence even in deep brain regions of live animals - with minimal invasiveness. Our setup will comprise an upright microscope fitted with technology to maintain and find again in successive sessions a specific position in the fly brain (PIFOC or AODs). It will display a large distance (20 cm) between objective and stage to allow fitting the fly, exposed to an olfactometer, reached by a feeder and positioned on a ball. It will display a tunable laser needed to unequivocally identify individual neurons, while recoding their responses to odor stimulation and to be prepared for future developments. We will perform volumetric scans with volumes of up to 50 microns. For this, we require a high speed of scanning (galvo and resonant scanners combined with PIFOC or acousto-optic deflectors). The functional signal detected in our preparation is a change in fluorescent intensity of a genetically encoded calcium indicator, associated with variations in calcium concentration. The signal level is inherently low and requires highly sensitive detectors. In addition, we utilize a second fluorescent structural marker to identify individual cells, realign the images over time and for motion correction. Therefore, the setup will include 2 highly sensitive photomultipliers with a good saturation protection system to avoid overload when encountering the highly reflective fly cuticle. The 25X objective will display high Numerical Aperture (N.A. 1.1) and a long working distance (2.0 mm). To locate the sample an epifluorescence microscope along the same light path is needed.
DFG Programme
Major Research Instrumentation
Major Instrumentation
2-Photonen Mikroskop
Instrumentation Group
5090 Spezialmikroskope
Applicant Institution
Rheinisch-Westfälische Technische Hochschule Aachen
