Method and apparatus for the determination of phase delay in a lifetime fluorometer without the use of lifetime standards

Abstract

A method for measuring the fluorescent lifetime of an unknown fluorescent sample, such as a biological liquid, without the requirement of manual intervention or a separate reference standard. An energetic light beam having its low energy component filtered out is shined on the sample. An optical detector is positioned such that the intensity of the components of the light beam reflected from the sample reaching the detector is substantially minimized but light remitted and/or fluoresced by the sample does reach the detector with non-trivial intensity. Errors arising from the electronic components of the system, such as RFI and D.C. offset errors, are isolated and minimized by positioning an opaque filter between the sample and the detector and measuring the resulting signal. The resulting baseline measurement data is stored for later error subtraction. A low-pass filter is selectively placed between the sample and the detector to isolate the high-energy remitted light component coming from the sample and having the same phase as the original energetic beam used to stimulate fluorescence, and a high-pass filter selectively is placed between the sample and the detector to isolate the lower-energy fluoresced light component coming from the sample. The phases of each component are then measured and compared to determine the phase shift of the light fluoresced by the sample, and the phase shift data is used to calculate the fluorescent lifetime of the sample according to tan &phgr;=&ohgr;&tgr;.