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

2. Green EmissionRed Emission 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 2

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

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

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

6. Imaging Capabilities for Membrane Receptors Single Molecule Determination of Subunit Stoichiometry TIRF-FRET (Sensitized Emission) High Resolution Localization Techniques (PALM, STORM) Quantitative FRET Single Photon Counting Single Receptor Ca 2+ Imaging for Functional Studies Simultaneous Correlation of Functional Studies, Stoichiometry, and FRET Current Developing 6

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

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