An improved instrument for measuring time-resolved lanthanide emission and resonance energy transfer

Abstract

Time-resolved fluorescence, particularly of millisecond lanthanide emission, is of considerable interest because of potentially increased sensitivity when short-lived autofluorescence is a problem. Lanthanides have also previously been shown to be excellent donors in resonance energy transfer measurements, where a lanthanide donor transfers energy to a conventional fluorophore. An instrument for the detection of micro- to millisecond lanthanide luminescence and delayed fluorescence, amidst a background of large prompt fluorescence, is described. Time-delayed emission spectra can be measured with 16 μs dead time while suppressing prompt fluorescence by 25 000-fold. This is accomplished by using a mechanical chopper placed very close to the entrance slit of a diffraction spectrometer, and a charge coupled device detector at the exit port. Excited-state lifetimes can also be measured with a 22 μs dead time while reducing detector ringing due to prompt fluorescence to an insignificant level. This is accomplished by high-voltage gating of a photon-counting photomultiplier tube in combination with a fast constant-fraction discriminator and amplifier. These improvements, which utilize all commercially available equipment, reduce the dead time by a factor of 10 for delayed emission measurements and a factor of 5 for lifetime measurements over a previously described instrument. The current instrument is particularly well suited for lanthanide-based resonance energy transfer measurements.