1. Dyes  Photostimulation: Channel Rhodopsin Caged glutamate (and others)  Ca 2+ sensitive  Voltage-Sensitive dyes  FRET, GRASP  Quantum Dots  Ultra-High-Resolution Light Microscopy

2. Channel Rhodopsin/Halorhodopsin

3. Channel Rhodopsin - structure Many thanks to Brikha!Gary G. Matthews, Neurobiology book

4. Channel Rhodopsin Switch

5. ChR2, NpHR pros/cons Pros Substitution for stimulating electrodes Stimulation of a great many cells at once Genetic targeting of cells subsets Less invasive compare to electrophysiology Cons Hard to get expression in a single cell Hard to get to the deep tissue (fiber-optics) Variable expression levels, hence unknown response

6. Glutamate uncaging Release glutamate upon illumination with 355nm (ultraviolet) light Temporal spike jitter: 1-3ns (10ns) Spatial resolution: ~5μm Other caged compounds: GABA, glycine ROPING (Reverse Optical Probing

7. Ca 2+ imaging Vogelstein et al, 2009 Calcium indicators Good SNR Saturation Slow Signal (decay ~2s) New statistical techniques for optical inference Paul De Koninck, 2007 !! VIDEO DEMO!!

8. Voltage-sensitive dyes Usually spans the membrane Dipoles orient themselves and change value ~ΔV For one cell in vertebrates : Low SNR But works great in Invertebrates! (Briggman, Kristan – leech, now – tritonia (>100 neurons, <1ms resolut.) www.umsl.edu/~tsytsarev/tsytsarev_files/Lecture10.htm

9. FRET (Förster Resonance Energy Transfer) sorry, no references for the figures

10. FRET (Förster Resonance Energy Transfer) Is extensively used in protein-protein interaction studies Occurs only when proteins get closer than 10nm Energy is transferred via induced dipoles of Fluorophores Formally the emission-absorption spectra can be used Carl Schoonover NSF proposal GRASP (GFP Reconstitution Across Synaptic Partners) - Evan H. Feinberg (Cori Bargmann)

11. Quantum Dots Nanoscale semiconductors The emission frequency depends on the particle size (the larger, the redder) Problem is how to deliver inside

12. Ultra-high-resolution light microscopy STED (Stimulated Emissions Depletion) / GSD (Ground State Depletion) - RESOLFT PALM (Photo-Activated Localization Microscopy) / FPALM (Fluorescent PALM) / STORM (STochastic Optical Reconstruction Microscopy) SI (Structures Illumination) (wide-field)