1. ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang

3. Design principles of caged compounds H. Lester and J. Nerbonne (1982) Ann. Rev. Biophys. Bioeng. 11:151

4. The dark reaction Decay of the aci-nitro intermediate of NPE-caged ATP J.W. Walker et al.(1988) JACS 110:7170

5. K.R. Delaney and R.S. Zucker (1990) J.Physiol. 426:473 Fast temporal control: caged calcium at the squid giant synapse

6. Delays in Ca release after IP 3 uncaging K. Khodakhah and D. Ogden (1993) PNAS 90:4976 Note: 1) [IP 3 ]-dependent delay in Ca rise and I K(Ca) ; 2) phosphorescence artifact Temporal dissection of signal kinetics

7. Judging a caged compound In practice, most caged compounds marketed have pretty fast dark reaction. A more variable quantity is the effectiveness with which caged compounds use light. The uncagability index depends on: Absorption (Tends to be constant for a given cage group) Quantum yield (Varies with modified molecule)

10. Focal uncaging Wang and Augustine (1995)

12. Two-photon excitation: the third dimension of resolution

13. Caged fluorescein dextran

14. Svoboda, Tank & Denk (1996) Science 272:716 Uncaging in single dendritic spines

15. Furuta et al. (1999) PNAS 96:1193

16. Comparison of a new caging group, 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), with previous caged compounds

17. Scanning two-photon uncaging of glutamate

19. Chemical two-photon uncaging Achieving a multiphoton effect by chemical means A new design principle: multiple-site caging Reduction of effective spontaneous hydrolysis Effective cross-section is MUCH larger (10 9 -fold) than true two-photon excitation

20. Chemical two-photon uncaging

22. Improved axial resolution via chemical two-photon uncaging

23. Wang, Khiroug and Augustine (2000) PNAS 97:8635

24. Wang, Khiroug and Augustine (2000) PNAS 97:8635

25. LTD induction causes a spreading decrease in receptor sensitivity

27. PART 2: TECHNICAL PRACTICALITIES

28. Handling caged compounds Regarding the necessity of keeping the compound in the dark. Storage. Vendor impurities - aftermarket purification. Cost control: recirculating and local perfusion.

29. Picking a caged compound Caged glutamates: a consumer report Fastest: CNB- or desyl- Best optical cross-section: Brc- Most efficient two-photon effect: bis-CNB- Future potential for two-photon uncaging: Corrie’s Magickal Indoline

31. Furuta et al. (1999) PNAS 96:1193

33. Nd:YAG 355 nm

35. Picking a light source If temporal only, light source can be uncollimated Flashlamps (Rapp) Mercury arc (Denk) Nd:YAG laser Argon laser Ti:S laser …see CSHL chapters by Delaney, Kandler

36. Achieving lateral resolution Full-field epi-illumination (>50 µm) Fiber optic directly into the preparation (20 µm) Epi-illumination with an aperture (5-50 µm) Focal beam direction (2-5 µm) - Ar laser or intense conventional UV source Diffraction-limited focus (<1 µm) - Ar or Ti:S laser Diffusion: another fundamental limit

39. How much light is enough? Light density Focal or subthreshold uncaging: 0.01-0.1 µJ/µm 2 Going through thick tissue may require more Photostimulation may require more

40. Alignment and focusing Light metering General focusing: fluorescence or caged fluorescein In epi-illumination mode, strive for parfocality With a UV objective, direct viewing is sufficient to achieve parfocality

41. Absorption bands imply chromatic aberration H. Piller, Microscope Photometry (1977)