Seeing is believing. To understand the brain and nervous system, we need to know how mechanisms are mediated at the subcellular, cellular, and network levels. Regulation of neuronal communication at synapses is an example of a complex response at the subcellular level, with impacts on neuronal network properties that in turn support functions from visual processing and pain regulation, to motor control, memory formation and emotion. Cellular signaling, homeostasis and change rely on proteins. In neurons, we need to be able to observe protein dynamics (activity, localization, molecular interactions) within the tiny compartment of the synapse, as they occur in the context of neuronal activity and synaptic signaling.
NORBRAIN UiB will provide three complementary platforms for nanoscale optical interrogation of neuronal and synaptic function. The platforms will enable: 1) super-resolution-based localization and tracking of protein movement in cell culture preparations, 2) imaging of protein-protein interactions in intact tissue such as rodent brain slices, and 3) structural and functional imaging of single neurons and networks in intact neural tissue, in combination with neurotransmitter uncaging to study structural and functional plasticity and dendritic integration.
Multiphoton FLIM-FRET
Many signal transduction events in cells are mediated by protein-protein or protein-RNA interactions. Changes in protein activity are often mediated by a change in protein conformation. Such interactions and conformational changes can be monitored by the fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores attached to the molecules of interest. The most reliable method for FRET detection in live cells is fluorescence lifetime imaging (FLIM). FLIM-FRET is based on detecting changes in fluorescence lifetime of the donor, and is independent of donor and acceptor concentrations in the cell.
This instrument will allow two-photon-based FLIM-FRET imaging in live thick-tissue specimens such as brain tissue slices. The methods requires expression of genetically-encoded fluorophores.
