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ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang. Artemis uncages the messenger. Physiol. Rev. (1987) 67:583. Light sensitive chymotrypsin inhibitor: an early ‘caged’ compound. Kaufman, Vratsanos & Erlanger (1968) Science 162:1487. Design principles of caged compounds.

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artemis uncages the messenger
Artemis uncages the messenger

Physiol. Rev. (1987) 67:583

light sensitive chymotrypsin inhibitor an early caged compound
Light sensitive chymotrypsin inhibitor:an early ‘caged’ compound

Kaufman, Vratsanos & Erlanger (1968)

Science 162:1487

design principles of caged compounds
Design principles of caged compounds

H. Lester and J. Nerbonne (1982)

Ann. Rev. Biophys. Bioeng. 11:151

the dark reaction
The dark reaction

Decay of the aci-nitro intermediate of NPE-caged ATP

J.W. Walker et al.(1988) JACS 110:7170

fast temporal control caged calcium at the squid giant synapse
Fast temporal control:caged calcium at the squid giant synapse

K.R. Delaney and R.S. Zucker (1990) J.Physiol. 426:473

temporal dissection of signal kinetics
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

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)

Judging a caged compound
absorption spectra of some caged nucleotides
Absorption spectra of some caged nucleotides

J. Nerbonne (1986) Design and application of photolabile intracellular probes. In: Optical methods in cell physiology, ed. P. De Weer and B.M. Salzberg.

focal uncaging
Focal uncaging

Wang and Augustine (1995)

uncaging in single dendritic spines
Uncaging in single dendritic spines

Svoboda, Tank & Denk (1996)

Science 272:716

slide21
Comparison of a new caging group,6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), with previous caged compounds
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 (109-fold) than true two-photon excitation

Chemical two-photon uncaging
double caged ip 3
Double-caged IP3

Goal: uncaging IP3 in single dendritic spines.

S.E. Gelber, J.W. Walker, S.S.-H. Wang

handling caged compounds
Regarding the necessity of keeping the compound in the dark.

Storage.

Vendor impurities - aftermarket purification.

Cost control: recirculating and local perfusion.

Handling caged compounds
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

Picking a caged compound
designing an uncaging setup
Picking a light source

Directing the light beam

Spatial resolution

How much light?

Alignment and calibration

Designing an uncaging setup
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

Picking a light source
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

Achieving lateral resolution
how much light is enough
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?
axial resolution the third dimension
High numerical aperture

Two-photon uncaging

Chemical two-photon uncaging

Axial resolution: the third dimension
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

Alignment and focusing
absorption bands imply chromatic aberration
Absorption bands imply chromatic aberration

H. Piller, Microscope Photometry (1977)

unwanted effects of uncaging
Light-induced toxicity

Photosensitization (radicals from the excited state?)

Photoproducts: photolyzed cage, H+ (toxic products?)

Controls: light alone; photolysis of cage alone

Unwanted effects of uncaging
quantitative calibration
General reporter: fluorescein

Caged phosphates: pH indicator

Caged calcium: Ca2+ indicator

Quantitative Calibration