Single photon photolysis of caged compounds provides temporal control of the stimulatory signal, but limited spatial control. A focused photolysis beam allows for increased spatial resolution, but still uncages biologically active species outside of the focal volume along the beam of illumination. MPE can be used to both spatially and temporally isolate the photorelease of a caged bioeffector such as a neurotransmitter or calcium. The figure at right demonstrates the spatial localization obtained using MPE uncaging. A DMNB-caged fluorescein containing polymer was scanned using pulsed 710 nm excitation resulting in photolysis only in the focal plane. This technique also has the potential to minimize out-of-focus photodamage since unfocused red light is more benign than UV. We have developed methods to measure the wavelength dependence of multiphoton photolysis of several caging groups.

CNB and DMNB caging groups The effective two photon cross sections of 4,5- dimethyloxy-2-nitrobenzyl ester (DMNB) and (alpha-2-carboxy)-2-nitrobenzyl (CNB) caging groups were measured (Zipfel et al, 1996). We used CNB-caged gamma-aminobutyric acid (CNB-GABA) for measurements of the properties of the CNB caging group, and DMNB-caged fluorescein as a representative compound for the DMNB group. Since the photolysis process is irreversible the method simply consists of controlled illumination and quantification of photo product. The two compounds were photolysed using a frequency doubled optical parametric oscillator pumped by a Ti:S laser (for CNB), or a Ti:S laser alone (for DMNB). Small aliquots (50 ml) of 25 mM caged compound was raster scanned avoid focal volume depletion. Photo released GABA was quantified using HPLC analysis, and released fluorescein by comparison with standards. The effective MPE cross-sections of these two caging groups are shown below:

	Calcium Cages Our technique for determining the effective two-photon cross sections of calcium cages requires a brief pulse train of high intensity focused laser light within a solution containing caged calcium and a fluorescent calcium indicator dye such as Fluo-3. The high intensity pulse train releases a fraction of the caged calcium, which is rapidly chelated by the fluorescent indicator dye. The same laser beam that uncages the calcium is attenuated after the photolysis pulse train and is used to generate fluorescence from the indicator dye before it diffuses out of the probe volume. The resultant fluorescence decay curve is monitored and fit to a model (Shear et al, 1996) yielding the effective uncaging cross sections of the cages and the diffusion coefficient of the indicator dye. Using this technique we have measured two commercially available cages, NP-EGTA and DM-Nitrophen, and a new calcium cage, Azid-1 (S. Adams and R. Tsien, UCSD). The effective uncaging cross sections of Azid-1 and DM-nitrophen are shown above. The cross-section of NP-EGTA was too small to yield detectable calcium levels at any of the wavelengths studied. Zipfel, W.R, Williams R.M. and W.W. Webb. Biophys J., 70(2):A196, 1996. Shear, J., Brown, E., Adams, S., Tsien, R., and W. Webb. Biophys. J. 70(2):A211, 1996.


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Last update: August 13, 2003

CNB and DMNB caging groups