Phase-modulation fluorometry using a frequency-doubled pulsed laser diode light source.

We used the Hamamatsu model PLP-01 picosecond light pulser as a 413-nm excitation light source for frequency-domain fluorescence measurements. In comparison with sync-pumped/cavity dumped/frequency-doubled dye lasers, the 413-nm PLP-01 shows a longer FWHM (40 ps), a similar pulse repetition rate (up to 10 MHz), much less output power at a fixed wavelength (0.44 mW peak, 220 nW maximum average power), but is less expensive, small-sized, and easy to handle. Using the PLP-01, we have been able to perform fluorescence measurements up to an upper modulation frequency of about 2000 MHz, and to resolve mixtures of fluorophores exhibiting different lifetimes. During our tests, we observed remarkable and lasting (2 h) time drifts between the optical output and the electrical trigger input or output. At present, work is in progress at Hamamatsu to eliminate these drifts.