Frequency domain fluorometry with pulsed light-emitting diodes.

We present a simple way to extend the time resolution of a standard frequency domain (FD) fluorometer by use of pulsed light-emitting diodes (LEDs) as an excitation source. High temporal resolution of the multifrequency FD method requires the excitation light to be modulated up to the highest possible frequencies with high modulation depth. We used harmonic content of subnanosecond-pulsed LEDs for generation of modulated excitation light. By a replacement of the light source, the upper frequency limit increased to 500-600 MHz, which is almost triple the frequency limit of the standard FD fluorometer equipped with an ordinary photomultiplier tube and an electro-optical modulator. Besides the increased time resolution, this approach allowed for elimination of a light modulator with an associated synthesizer and radio frequency power amplifier that are normally required for FD measurements with continuous wave light sources. Performance of the instrument with pulsed LED excitation is demonstrated on several examples of ultraviolet-excited fluorescence decays. We show that pulsed LEDs can serve as an inexpensive alternative to pulsed laser sources for FD fluorescence spectroscopy.