Novel fluorescence sensing methods for high throughput screening

We describe two new methods of fluorescence sensing for use in high through put screening (HTS). Modulation sensing transforms analyte-dependent intens ity changes into a change in the low-frequency modulation signal. Polarizat ion sensing transforms an intensity change into a change in polarization. B oth methods are internally calibrated by using a reference film immediately adjacent to the sample, which can be readily located on the HTS plate or o n a nearby optical component and provides an intensity or polarization refe rence. Modulation sensing and polarization sensing were both shown useful f or measurements of fluorophore concentrations, pH, or calcium concentration s in the wells of HTS plates. Sensing with a reference film provides the op portunity to internally reference HTS measurements without the need for add itions to the sample. This approach can provide standardization for assays performed at different times.