Systems and methods for single molecule quantification

A method of determining an amount of individual optically active dye labels immobilized on a substrate, the method comprising:

• at an electronic device with one or more processors and memory:
• (a) obtaining digital image data corresponding to an image of the substrate, wherein the digital image data comprises an array of intensity values, wherein each intensity value of the array is associated with a location on the substrate;
• (b) segmenting the digital image data into a plurality of regions, each comprising an intensity value that is above a predefined threshold;
• (c) for each of the plurality of regions:
• (i) providing a count of one optically active dye label;
• and
• (ii) subtracting a reference signal distribution from the intensity value to obtain one or more adjusted intensity values, wherein the reference signal distribution corresponds to a point spread function of a single optically active dye label;
• (iii) identifying one or more of the adjusted intensity values comprising a value above the predefined threshold;
• and
• (iv) increasing the count by one for each of the one or more adjusted intensity values identified in (iii).