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NROC69H3 (7)

Lec 2 STED Paper main points

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Daman Bawa

Neurochemical transmission •NT released by exocitosis •Vesicle membrane retrieved by endocytosis -> vesicle regenerate and refill Synaptic vesicles ~40nm diameter and densely packed Synaptotagmin I - protein resident in the vesicle membrane MODEL OF NT RELEASE Full fusion •Synaptic vesicle membrane undergoes exocytosis by collapsing into the plasma membrane •Involves mixing and lateral diffusion of vesicle proteins across plasma memb before they are re-internalized by endocytosis -proteins need to be sorted at plasma memb and or in an endosomal intermediate to regenerate vesicle -if vesicles constituents stay patched together after exocytosis, the recycling machinery would just have to internalize the fused vesicular patch without sorting STED THEORY STED - stimulated emission depletion •Resolve individual vesicles in synapse •Breaks the diffraction barrier of far-field fluorescence microscopy •Allows diffraction-unlimited resolution despite usage of regular lenses •Typical STED microscope, the excitation beam is overlapped with a doughnut-shaped beam that is capable of de-exciting fluorophores by stimulated emission •Co-alignment of the beams ensures that fluorescence is allowed only in the central area of the excitation spot where the doughnut beam is close to 0 •Scanning w a narrowed spot across the sample yields subdiffraction images STED SETUP •STED microscope: objective lens 1.4 numerical aperture •The green-emitting dye used for antibody labelling excited at 470 nm, de-excited at 615 nm •Doughnut setup yielded a spot FWHM of 66nm -> 9x reduction in effective focal area -spot of STED microscope thinner towards top -> resolution slightly better •Separated point objects 45nm apart in focal plane VESICLE LABELLING •Vesicles targeted w a monoclonal antibody directed against intravesicular domain of synaptotagmin •When neuron active, a vesicle opens to outside space (exocitosis), rendering its inside accessible to antibodies -> bind to synaptotagmin molecules -> internalized when vesicles are retrieved • Only vesicles undergoing exocytosis during incubation are labelled • Visualized by fluorescently labelled secondary antibodies applied after membrane pe
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