1 / 42

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

ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang. Design principles of caged compounds. H. Lester and J. Nerbonne (1982) Ann. Rev. Biophys. Bioeng. 11:151. The dark reaction. Decay of the aci -nitro intermediate of NPE-caged ATP J.W. Walker et al. (1988) JACS 110:7170.

kareem
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDSSamuel S.-H. Wang

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

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

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

  5. Temporal dissection of signal kinetics Delays in Ca release after IP3 uncaging K. Khodakhah and D. Ogden (1993) PNAS 90:4976 Note: 1) [IP3]-dependent delay in Ca rise and IK(Ca); 2) phosphorescence artifact

  6. 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) Judging a caged compound

  7. Focal uncaging Wang and Augustine (1995)

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

  9. Caged fluorescein dextran

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

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

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

  13. Scanning two-photon uncaging of glutamate

  14. 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 (109-fold) than true two-photon excitation Chemical two-photon uncaging

  15. Chemical two-photon uncaging

  16. Improved axial resolution viachemical two-photon uncaging

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

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

  19. LTD induction causes a spreading decrease in receptor sensitivity

  20. PART 2:TECHNICAL PRACTICALITIES

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

  22. 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 Picking a caged compound

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

  24. Nd:YAG 355 nm

  25. 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 Picking a light source

  26. 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 Achieving lateral resolution

  27. Light density Focal or subthreshold uncaging: 0.01-0.1 µJ/µm2 Going through thick tissue may require more Photostimulation may require more How much light is enough?

  28. 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 Alignment and focusing

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

More Related