FREQUENCY-DOMAIN FLUORESCENCE SPECTROSCOPY - INSTRUMENTATION AND APPLICATIONS

Time-resolved fluorescence measurements are often performed by direct measurements in the time-domain. In this article we describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments operating with picosecond laser systems and fast detectors, which allow measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article we describe this instrumentation and applications of the frequency-domain method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of diffusion dependent energy transfer. We also describe the use of a pulsed frequency-doubled laser diode, as a 413 nm excitation light source for frequency-domain fluorescence measurements.