We developed an image-to-answer algorithm available for chip-based dPCR systems with single fluorescence image capture and known image-related error removal. It automatically corrects the chip structure damage and non-uniformity of light intensity.

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (T_M). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured T_M values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured T_M as the parameter. Even when the actual T_M of the PCR solution was constant, the measured T_M value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known T_M as a temperature sensor. Among the non-contact temperature measurement methods, the proposed T_M-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on T_M allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.

Fluorescence imaging underpins digital PCR (dPCR), microarrays, and microfluidic biosensors, yet precise image integration remains a technical bottleneck when the sample area exceeds the microscope field of view.