Multiplex analysis of molecules in single cells by image correlation

A method of determining an abundance level of at least two different molecular targets within a defined region in a cell, comprising:

• (a) providing a sample comprising the cell;
• (b) carrying out a plurality of {1, 2, . . . N} hybridizations on the sample, wherein N is equal or greater than 2, and wherein the plurality of hybridizations interacts with the at least two molecular targets, and wherein each hybridization is performed on the sample by applying a hybridization-specific plurality of probes to the cell, wherein each plurality of probes comprises at least two probes, one probe interacting with at least one of the two different molecular targets, and the other probe interacting with at least the other of the two different molecular targets;
• (c) imaging the sample after each hybridization and obtaining an image produced by the interaction between the at least two probes of that hybridization and the at least two molecular targets, wherein the image at least partially constitutes visual data for the hybridization, and wherein at least one image per fluorophore channel per hybridization is taken;
• (d) identifying, in the defined region in the cell, an abundance of the at least two molecular targets by using a correlation function, wherein the correlation function spatially correlates visual data from two or more images;
• and
• (e) determining an abundance level of the at least two molecular targets based on the correlation function.