Hans Anderson, Richard Weir, and John Caldwell of the University of Colorado have developed a completely automated method for the quantification of protein expression in immuno-labeled cells. Developed initially to determine transgene expression in peripheral motor axons, this method utilizes three spectrally-separated images; one of which is utilized as a negative mask, and one as a positive mask, and the third image to identify the target using colocalization techniques. The software then leverages ImageJ and the Nucleus Counter plugin to do a high throughput analysis of the images to determine protein expression with an accuracy of 95%. This approach overcomes the problems associated with manual and semi-automatic methods which are time consuming to implement and can also suffer from inter-observer, intra-observer, and sampling bias.