Figure 3.

PCR thermal profile of a single amplification step. The heater temperature (red part) is linearly proportional to the built-in lock-in amplification output, which was captured with an oscilloscope. While performing denaturation at ≈ 93 °C, annealing at ≈ 56 °C and most of the extension step at ≈ 72 °C, the built-in lock-in amplifier is utilized to measure an average temperature of all four heaters. In the last two seconds of the extension step, the lock-in amplifier is used to sequentially process the fluorescence signal (green part) from the 4 measurement spots (circled area). These data are stored in their original format in analog-to-digital converter units (ADC units) within the memory of a microcontroller. Once fluorescence is measured, the heater is powered by an average value of pulse width modulation obtained during the extension phase.