Fluorescence resonance energy transfer (FRET) was being widely used in the eighties and nineties to measure intermolecular distances, using either natural donor and acceptor fluorophores already present within biomacromolecules, or using fluorophores covalently adducted to such molecules as additional molecular beacons, or labels. The problem was that most groups were measuring FRET efficiency through steady state measurements of the apparent drop in donor quantum yields, using ordinary fluorimeters, rather than through measurements of the drop in the fluorescence lifetime of the donor, using instruments measuring time-resolved fluorescence through pulsed excitation and single photon counting and/or box-car measurement methods. While I was attempting to do some FRET work with lens crystallins, it struck me that a drop in the donor’s lifetime would accurately provide a measure of FRET efficiency but that a drop in the donor’s emission intensity could result either from FRET or from simple (trivial) reabsorption of donor-emitted photons by acceptor molecules in solution, on their way to the instrument’s detector, masquerading as FRET and contaminating all steady-state illumination-based measurements of FRET. In this paper, I and a colleague from the lab in which I did a Ph.D (B. Raman) demonstrate the reality of such trivial reabsorption which masquerades as FRET, and contaminates FRET measurements, through the use of a ‘tandem’ quartz cuvette composed of two compartments separated by a 0.5 mm thick wall of quartz, to show that separated donor and acceptor fluor-containing solutions in the two compartments can display apparent FRET. All arguments against the use of steady-state illumination and favouring lifetime measurements for FRET studies are also included in considerable detail, in the discussions. I can guarantee that anyone who reads and understands this paper will never again use steady-state illumination for FRET measurements; indeed, the standard textbook by Lakowicz on fluorescence spectroscopy includes a reference to this paper in its chapter on FRET, in its sixth and subsequent editions.

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