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

We describe the use of a pulsed frequency-doubled laser diode, the Hamamatsu model PLP-01 picosecond light pulser, as a 413-nm excitation light source for frequency-domain fluorescence measurements. The modulated incident light, over a range of modulation frequencies, is provided by the harmonic content of the pulse train. In comparison with the more usual light source for harmonic-content excitation, a sync-pumped/cavity dumped/frequency doubled dye laser, the 413-nm PLP-01 has a longer pulse width (FWHM of 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 were able to perform frequency-domain 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.