Abstract
Advances in laser technology and probe chemistry are resulting in a rapid introduction of novel fluorescence measurements. One area of rapid growth has been multi-photon excitation, which is now practical due to the increasing availability of ps and fs lasers. The interest in multi-photon excitation is driven in part by the possibility of three-dimensional “confocal” cellular imaging based on the localized excitation possible with multi-photon excitation. In this paper we will show that using the fundamental output of a fs Titanium:Sapphire laser it is possible and practical to observe three-photon excitation of proteins, DNA stains, calcium probes, and labeled membranes.
Most studies of two-photon excitation use two photons of the same wavelength. We recently demonstrated that two-photon excitation can be obtained using two-photons at different wavelengths. The potential advantages of two-color two-photon excitation include localization of the excited volume at the region of beam overlap, and the possibility of increased selectivity by independent control of each laser beam.
During the past several years we developed a new method to control of the excited state population using laser pulses. The present availability of multi-wavelength laser sources allows the use of stimulated emission to quench and modify the excited state populations. We refer to this method as light quenching, which allows selective removal of excited state fluorophores based on emission wavelength, decay time or orientation. In the case of evanescent wave excitation due to total internal reflection (TIR) we show that light quenching can selectively remove fluorophores from the interface region, and provide spatially localized excitation 5000 Å into the aqueous phase.
And finally, we describe the development of metal-ligand complex (MLC) probes which provide the opportunity to measure dynamics on the microsecond timescale. This versatile class of fluorophores allows a wide range of decay times and emission wavelengths based on the choice of ligand and metal. Importantly, luminescence lifetimes can be as long as 10 µs. Many MLC probes display high fundamental anisotropies. A recently synthesized rhenium-MLC displays a decay time of 3 microseconds in oxygenated aqueous solution, and a still longer decay time in the absence of oxygen. Conjugatable MLCs have already been developed, as well as a pH- sensitive MLC with a pKa near 7.4. These probes are also expected to enable optical clinical chemistry using lifetime-based sensing with low cost LED-based instrumentation.
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Lakowicz, J.R. et al. (1998). Recent Developments in Fluorescence Spectroscopy. In: Daehne, S., Resch-Genger, U., Wolfbeis, O.S. (eds) Near-Infrared Dyes for High Technology Applications. NATO ASI Series, vol 52. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-5102-3_1
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DOI: https://doi.org/10.1007/978-94-011-5102-3_1
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