A new method for filter-free fluorescence measurements

Fluorescence measurements usually evaluate the intensity and the decay- time of the fluorophore. For the suppression of the excitation light at the optical receiver optical filters are generally used. In this paper, a technique is described which provides the suppression of the excitation light component by electronic means, thus, eliminating the need for an optical filter. An intensity-modulated excitation light causes an intensity-modulated fluorescence signal. This fluorescence signal is mixed with a signal having a 90 degrees phase shift with respect to the excitation light. The product of two orthogonal signals equals zero and in this way the excitation light is suppressed. Measurements at two or more modulation frequencies make it possible to compute the relative intensity and the decay-time. The advantage of this technique is its flexibility in the adaptation to different fluorescent materials and its compact realization. This method was evaluated for Lucigenin quenched by chloride ions using an experimental set-up consisting of a LED, a photo multiplier, no optical filters and either a network analyzer (NWA) or a self-made electronic circuit. Although up to 10 times more primary light than fluorescence light was detected at the optical receiver, the omission of the optical filter did not contribute to undesired effects. The results were compared with measurements on the same samples using a conventional test set-up. There was no difference in the computed Stern-Volmer constant. Therefore, the new method offers a cost-effective but still exact alternative for measuring fluorescence intensity and decay-time down to the range of nano-seconds.