Fluorescent imaging of pH with optical sensors using time domain dual lifetime referencing

We present a referenced scheme for fluorescence intensity measurements that is useful for imaging applications It is based on the conversion of the fl uorescence intensity information into a time-dependent parameter. A phospho rescent dye is added in the form of similar to 10-mum particles to the samp le containing the pH-sensitive fluorescent indicator. Both the reference dy e and the pH probe are excited simultaneously by a blue LED, and an overall luminescence is measured. In the time-resolved imaging method presented he re, two images taken at different time gates were recorded using a CCD came ra. The first image is recorded during excitation and reflects the luminesc ence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measured after 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 the information on pH, wh ereas phosphorescence is pH-independent, the ratio of the images displays a referenced intensity distribution that reflects the pH at each picture ele ment (pixel). The scheme is useful for LED light sources and CCD cameras th at can be gated with square pulses in the microsecond range. The fundamenta ls and potential of this new method, to which we refer-as time domain dual lifetime referencing (t-DLR), are demonstrated.