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Multiphoton Voltage Imaging

Project description

Three could be the magic number when it comes to photons for imaging action potentials

The brain is a complicated electrical circuit that utilises voltage changes across cellular membranes as a key mediator of signalling. Numerous techniques have been used to explore the electrophysiology of brain circuits, from electrode recordings to the use of voltage-sensitive dyes. Genetically encoded voltage indicators (GEVIs) are an emerging technology for monitoring electrical dynamics. These engineered light-emitting protein sensors change their brightness in response to changes in neuronal voltage. The EU-funded MULTIVIsion project is advancing the capabilities of GEVIs with three-photon imaging. Three-photon imaging relies on the simultaneous interaction of three photons with a fluorescent molecule, exciting it to a higher electronic state than two-photon or one-photon imaging. The longer wavelengths used result in less scatter and clearer images, even in deeper tissues. A clearer 'snapshot' of an action potential could shed light on learning and memory formation at the level of individual neurons.


Understanding the brain is one of the great scientific challenges of our time. This pursuit fundamentally depends on advances in physical sciences and engineering to provide novel tools and methods to perturb, record and interpret brain activity.
Information in the brain is encoded in changes in the voltage across the membrane of brain cells. Voltage imaging with genetically encoded voltage indicators (GEVIs) is a revolutionary method that allows faithful recording of the fast electrical dynamics of many genetically targeted cells in parallel. This provides an unprecedented means to record how patterns of change in this membrane voltage, called action potentials, manifest in subcellular compartments, cells, and networks across the brain, which is the only way to arrive at a fundamental understanding of brain functions like learning and memory, and of neurogenerative diseases. For this promise to be fulfilled, we need voltage imaging deep in the living brain of awake and behaving organisms. To achieve this, I will evolve a GEVI optimized for three-photon (3P) imaging, by screening mutant libraries of GEVIs directly for brightness and photostability under 3P-excitation, voltage sensitivity, and membrane trafficking in neurons. I will optimize the photocycle dynamics of GEVIs with temporally structured light, using a NOPA with tunable repetition rate, pockels cell and multiple optical delay lines, to create optimal 3P excitation protocols. Crucially, this optimization will not depend on spectral phase and is therefore compatible with high speed multiphoton imaging, aberration correction, and deep tissue imaging. Finally, I will investigate memory formation in a proof-of-principle experiment. I will use a custom microscope I developed, for 3P voltage imaging of cells in the cerebellum of mice trained in an eyeblink task. Memory formation in this paradigm is hypothesized to look like subtle changes in the exact shape and timing of action potentials, which I will resolve.

Host institution

Net EU contribution
€ 1 499 655,00
2628 CN Delft

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West-Nederland Zuid-Holland Delft en Westland
Activity type
Higher or Secondary Education Establishments
Total cost
€ 1 499 655,00

Beneficiaries (1)