Systems and methods are provided for calibrating emission data or other information signals collected during a polymerase chain reaction (PCR), amplification reaction, assay, process, or other reaction. Calibration of multiple detectable materials can be achieved during a single cycle or run, or during a plurality of runs of the reaction. A reading from every well, container, or other support region of a sample support does not have to be taken. Interpolation can be used to determine values for emission data or other information signals that were not taken, or are unknown, using detected emission data, or other detected information signals. By calibrating the detected emission data and the interpolated data, a more accurate reading of emission data or information signal can be obtained.