Partition-free digital PCR (dPCR) system

A method for diffusion-controlled quantification of molecules comprising the steps of:

• introducing a sample into a chamber;
• wherein the sample comprises primers, amplification reagents, and one or more nucleic acids, and wherein the nucleic acids are distributed across the chamber;
• providing a thermal system in thermal communication with the sample;
• providing an optical system in optical communication with the sample comprising one or more nucleic acids, wherein the optical detection system comprises an optical sensor;
• performing amplification of the one or more nucleic acids;
• actively controlling the diffusion of the amplified sample within the chamber such that an amplification spot is formed at each location of a nucleic acid that underwent amplification;
• obtaining one or more images of the amplification spots;
• digitally quantifying the amplified nucleic acids based on fluorescence distribution of the amplification spots across the chamber;
• wherein, digitally quantifying the amplified nucleic acids comprises:
• analyzing the one or more images to determine full-width-half-maximum (FWHM) cluster boundaries that are indicative of single amplification spots;
• counting positive and negative spots based on the FWHM cluster boundaries, and,
• using a ratio of the positive and negative spots in the chamber to estimate quantification (λ).