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Fluorophore
Photostability & Properties of Caging Groups under MPE
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| Characterization
of Fluorophore Probe Photostability is
critical, because it limits both the retrieval rate and the total
quantity of fluorescence information that can be extracted from a
specimen. We have developed a reproducible and potentially routine
method for quantification of fluorophore photobleaching quantum yields
based on a commercially available laser scanning apparatus. In a uniformly
distributed sample the scanned beam illuminates a constant number
of fluorophores at any time. However at slower scan speeds it dwells
longer on certain fluorophores, imparting on them a higher photobleaching
probability. The method is applicable for both linear and nonlinear
excitation and is inherently consistent with experimental conditions
under which laser scanning microscopy is performed. The graph to the
right shows a plot of fluorescence attenuation as a function of scan
speed for fluorescein in an air-saturated aqueous environment. The
data indicate that for every 2000 molecules excited with 780 nm MPE,
one photobleaches. PROPERTIES OF CAGING GROUPS UNDER MPE 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 below 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. |
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Last
update:
August 13, 2003