Image based correction for unwanted light signals in a specific region of interest

All of these sources of variation can contribute to a dynamically changin...

[0040] After the region of correction calibration is performed, the correction based upon from the region of correction can be applied by using the following method. Once the region of correction calibration is performed, a background offset value needs to be generated. This value can be generated in at least two ways. According to a first alternative, a background calibration can be performed. In this method, an image is taken of a plate without any fluorescent dye. During the background calibration, the average pixel value for each region of correction is calculated by dividing the pixel sum by the pixel count in that region of correction to obtain an average pixel value. This average pixel value is indicative of the background light level and is referred to as the background offset value. The background offset values are stored for use in future runs, e.g., PCR runs. Alternatively, the background offset value can be determined on a run-by-run basis by calculating the average pixel value for each region of correction for the first reading of a run, e.g., a PCR run. Because the first (or first few) readings of a PCR run occur before a significant reaction signal is produced, this alternative method provides a good representation of the background.

[0041] The signal correction is performed in the following manner. Performance of signal correction is depicted in FIGS. 1 and 3. For each reaction, the reaction pixel sum and the reaction pixel count are calculated by using the reaction region of interest. The average pixel value for the four regions of correction associated with a given reaction region of interest is calculated. Although four regions of corr...

[0019] The method of this invention can be used to measure a dynamically changing signal and the effect of the dynamically changing signal on a region of interest of a specific reaction. Correcting for the cross-talk inherent in a dynamically changing signal will greatly increase the sensitivity of the method of detection used in an assay emp...