In January of 1987 we received NIH funds to construct a state-of- the-art instrument for time-correlated single photon counting fluorescence spectroscopy, which is now operational and is in constant use. The NIH funds provided a high repetition rate psec light source. This source is an Antares mode-locked Nd-YAG laser, a cavity dumped R6G dye laser, and a frequency doubler to provide wavelengths from 280-305 nm. The timing and data acquisition circuits are based on Model 199 from Edinburgh instruments, which was purchased with NIH shared instrument funds in 1984. A unique opportunity exists to make dual use of the laser light source for both time and frequency-domain fluorescence. As the result of recent developments it is now possible to use the same pulsed laser source for frequency-domain fluorometry. This is accomplished using the harmonic content of the pulse train, which extends to many GHz. The first such instrument allows measurements to 2 GHz with very good signal-to-noise (Lakowicz et al., Rev. Sci. Instrum. (1986) 57, 2599-2506), and the proposed instrument will allow measurements to at least 6 GHz and possibly as high as 10 GHz. The basic idea of this proposal is to allow both time and frequency-domain measurements to be performed on the same sample, using the same light source. This addition will provide Drs. Bucci, Steiner, Inesi, Jain and other users with access to GHz frequency-domain data and to all the software developed by Dr. Lakowicz. Shared use of the Nd-YAG source is operationally simple. If the same wavelength is to be used for measurement in both domains, then the output of a single dye laser will be shared using a beam splitter. Based on past experience we know there is adequate light for both experiments. Alternatively, the Antares Nd-YAG can pump two dye lasers, each with a different dye, because its output at 532 nm is 2 watts. Relatively little equipment is needed to implement the dual domain instrument. The needed items include a frequency synthesizer, MCP PMT, dye laser and doubler, reference photodiode, beam splitters and a inexpensive computer for data acquisition. The extensive frequency-domain software for acquisition and data analysis will be transferable to the shared- use instrument. Also, software for simultaneous analysis of time and frequency-domain data, from the same sample, will be available in two months.
| Pinsky, B G; Ladasky, J J; Lakowicz, J R et al. (1993) Phase-resolved fluorescence lifetime measurements for flow cytometry. Cytometry 14:123-35 |
| Lakowicz, J R; Gryczynski, I (1993) Characterization of p-bis(O-methylstyryl)benzene as a lifetime and anisotropy decay standard for two-photon induced fluorescence. Biophys Chem 47:1-7 |
| Lakowicz, J R; Gryczynski, I (1992) Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one-photon and two-photon excitation. Biophys Chem 45:1-6 |
