| Abstract
| - We present a referenced scheme for fluorescence intensitymeasurements that is useful for imaging applications. Itis based on the conversion of the fluorescence intensityinformation into a time-dependent parameter. A phosphorescent dye is added in the form of ∼10-μm particlesto the sample containing the pH-sensitive fluorescentindicator. Both the reference dye and the pH probe areexcited simultaneously by a blue LED, and an overallluminescence is measured. In the time-resolved imagingmethod presented here, two images taken at different timegates were recorded using a CCD camera. The first imageis recorded during excitation and reflects the luminescence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measuredafter a certain delay (after switching off the light source),is solely caused by the long-lived phosphorescent dye.Because the intensity of the fluorophore contains theinformation on pH, whereas phosphorescence is pH-independent, the ratio of the images displays a referencedintensity distribution that reflects the pH at each pictureelement (pixel). The scheme is useful for LED lightsources and CCD cameras that can be gated with squarepulses in the microsecond range. The fundamentals andpotential of this new method, to which we refer as timedomain dual lifetime referencing (t-DLR), are demonstrated.
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