Project description
Fast and precise shots of synaptic activity
Incompatible optical and probe requirements in current technologies are blocking studies of fast dynamics with intra-synaptic precision. This is a major challenge for the real-time functional imaging of brain activities. The EU-funded InSpIRe project will address this challenge. It will develop an optical platform that allows real-time volumetric imaging of brain tissues with an intra-synaptic level of details thanks to a new optical scheme coupled to the photoswitching properties of recently engineered red-shifted probes. It will take advantage of rsFusionRed, a new palette of reversibly switching fluorescent proteins. As a proof of principle, it will study the dynamics of the endoplasmic reticulum, which are little known in synapses, and could show these with precision.
Objective
Modern fluorescence microscopy can provide rapid, real-time functional imaging of brain activities (light sheet) or to visualize with high definition and specificity the synaptic organization (super resolution, expansion). However, rapidity and precision are still too far apart to allow studies of fast dynamics with intra-synaptic precision mainly due to incompatible optical and probes requirements in current technologies. My aim is to fill this gap with the development of an optical platform, named InSpIRe, which allows real-time volumetric imaging of brain tissues with intra-synaptic level of details thanks to a new optical scheme coupled to the photo-switching properties of recently engineered red-shifted probes. InSpIRe will take advantage of rsFusionRed, a new palette of reversibly switching fluorescent proteins, which we recently introduced. The contrast and photo-resistance of rsFusionRed imaging will be increased with an optical strategy that uncouple the geometry of the illumination for switching and fluorescence excitation, which we demonstrated in our MoNaLISA microscope. In InSpIRe these concepts are brought to a new level to record volumetric dynamics in brain tissues without compromising resolution and speed. We will craft a new interference pattern, which optically imprint small-sub-resolved- volumes of rsFusionRed in the 3D tissues architecture. These volumes will be read-out with an oblique sheet of light to increase speed and minimize photo-bleaching. Unlike lattice light sheet we use one objective lens, which increases the slice accessibility and unlike STED/STORM the acquisition is faster. InSpIRe will record movies with molecular resolution without losing the larger neuronal architecture, which can shine light to open question in the field of organelles trafficking. As a proof of principle, we will study the dynamics of the endoplasmic reticulum, which are little known in synapses, and which we could, for the first time, show with precision.
Fields of science
Programme(s)
Funding Scheme
ERC-COG - Consolidator GrantHost institution
100 44 Stockholm
Sweden