Method for deconvolving single-molecule intensity distributions for quantitative biological measurements

A method for quantifying fluorescent puncta, comprising:

• (a) acquiring at least one first intensity distribution, wherein the first intensity distribution comprises fluorescence intensity values from a plurality of first fluorescent puncta, and wherein each first fluorescent puncta comprises at least one fluorescent emitter;
• (b) acquiring at least one second intensity distribution, wherein the second intensity distribution comprises fluorescence intensity values from a plurality of second fluorescent puncta, wherein each second fluorescent puncta has a determined number of fluorescent emitters;
• (c) determining a statistical relationship between a shape of the first intensity distribution and a shape of the second intensity distribution;
• and
• (d) fitting the second intensity distribution to the first intensity distribution to provide a count and distribution of the number of fluorescent emitters within the first fluorescent puncta.