Spatially resolved ligand-receptor binding assays

A method for determining the concentration of a ligand in a sample comprising the steps of:

• (a) combining in a reaction mixture (i) a sample suspected of containing a ligand, (ii) a first receptor attached to a microparticle that binds to the ligand, and (iii) a fluorescently labeled second receptor that binds to the ligand, and allowing formation of a complex comprising the microparticle attached to the first receptor, the ligand, and the fluorescently labeled second receptor;
• (b) acquiring a white light image of the reaction mixture in order to determine the location of the microparticle in the reaction of step (a) and a fluorescence image of the reaction mixture in order to determine the location of the fluorescently labeled second receptor in the reaction of step (a);
• (c) selecting at least one region of interest from the images acquired in step (b), wherein the at least one region of interest is a region from which light signals emanate from the complex formed in step (a);
• (d) selecting pixels in the at least one region of interest for analysis;
• (e) calculating and recording the average and variance of the counts per pixel for the pixels selected in step (d), wherein the counts per pixel is the number of photons counted per pixel per unit of time;
• (f) omitting pixels that have counts greater or less than a specified variance;
• (g) calculating average counts per pixel of the remaining pixels;
• and
• (h) determining the concentration of the ligand from the data in step (g).