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Unformatted text preview: Frequency Domain Fluorometry with Pulsed Light-Emitting Diodes P ETR H ERMAN AND J AROSLAV V ECER Faculty of Mathematics and Physics, Charles University, Institute of Physics, Prague, Czech Republic 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 500600 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. Key words: harmonic content; UV LED; light-emitting diode; phase and modulation fluorometry; fluorescence; NADH; HSA; POPOP; fluorescence lifetime; Trp fluorescence Introduction Besides time domain measurements, frequency do- main (FD) fluorescence techniques are commonly used in fluorescence spectroscopy, fluorescence sensing, and fluorescence lifetime imaging. FD measurements rely on a periodically modulated excitation that forces fluo- rophores to emit on the same angular frequency. A nonzero fluorescence lifetime of the sample causes the emission to become phase shifted (delayed) relative to the excitation light, and the modulation depth of the emitted light decreases. Excited-state lifetime then can be calculated from the measured values of the phase shift and the demodulation ratio. Importantly, resolution of complex emission decays requires mea- surements on multiple modulation frequencies, and a temporal resolution of the method is related to the highest available frequency. Detailed description of the FD technique can be found in Lakowicz. 1 Phase methods attracted the attention of scientists mainly because of a rapid determination of fluores- Address for correspondence: Prof. Petr Herman, Ph.D., Faculty of Mathematics and Physics, Charles University, Institute of Physics, Ke Karlovu 5, 121 16 Prague 2, Czech Republic. Voice: + 420-221911461; fax: + 420-224922797....
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