Overcoming Autofluorescence (AF) and Tissue Variation in Image Analysis of In Situ Hybridization.

Fluorescent detection of nucleic acid sequences such as DNA or RNA allows for multiplexing and visualization of an increased number of targets compared with chromogenic methods. This is due to the number of chromogens available as well as the ability of image analysis software platforms to distinguish between colors. Autofluorescence (AF) can be problematic during fluorescent imaging because the AF interferes with the detection of the specific fluorescent signals especially when the target signals are weak. AF has a broad emission spectrum leading to difficulty when performing image analysis due to masking of the specific signal across multiple wavelengths. Tissue sample variation can also affect levels of AF. In this chapter we share a method for overcoming the issues caused by sample variation and AF using HALO software on RNAscope in situ hybridization images.