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Nicotinic Receptors at the Membrane

Nicotinic Receptors at the Membrane. Chris Richards Lester Group 01/15/2010. Single Molecule and Single Vesicle Imaging at the Membrane. N2a cells transfected with α 4-mCherry and β 2-GFP TIRF Imaging Receptors and Vesicles Arriving at the Membrane. Green Emission. Red Emission.

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Nicotinic Receptors at the Membrane

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  1. Nicotinic Receptors at the Membrane Chris Richards Lester Group 01/15/2010

  2. Single Molecule and Single Vesicle Imaging at the Membrane N2a cells transfected with α4-mCherry and β2-GFP TIRF Imaging Receptors and Vesicles Arriving at the Membrane Green Emission Red Emission

  3. Single Molecule Time Trace Subunit Counting Determine Stoichiometry Membrane Dwell Time

  4. SynaptopHluorin in N2a Cells pH Sensitive GFP – Synaptobrevin Dark on the Luminal Side of the Vesicle Activated on Membrane Insertion Monitor Vesicle Trafficking and Endocytosis Clear Arrival Events Almost no Endosomal Like Activity

  5. Merge of nAChR-mC and s-pH Channels Cotransfected nAChRs and SynaptopHluorin Arrival Events Endosomal Activity No Clear Overlap Between mC and S-pH Channels

  6. Imaging Capabilities for Membrane Receptors Current • Single Molecule Determination of Subunit Stoichiometry • TIRF-FRET (Sensitized Emission) • High Resolution Localization Techniques (PALM, STORM) • Quantitative FRET • Single Photon Counting • Single Receptor Ca2+ Imaging for Functional Studies • Simultaneous Correlation of Functional Studies, Stoichiometry, and FRET Developing

  7. Current Focus Incorporation of Ecliptic pHluorin Incorporation of Halo Tag Optimization of Single Molecule – Turnover Experiments Single Molecules and Vesicles in Neurons

  8. Building Toward a Multi-functional Imaging Platform Sample Piezo Stage BS S1 M1 488 nm 514 nm 561 nm M2 CCD 1 S2 L2 TL Opto Split L1 DM DM M2 FM Ls1 CCD 2 Ms1 DM PH S3 Ms2 AOTF DM APD 1 633 nm Router TCSPCM APD 2 Widefield/Confocal Path TIRF Path Secondary Laser Path

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