||Image-based sequencing of RNA molecules directly in tissue samples provides a unique way of relating spatially varying gene expression to tissue morphology. Despite the fact that tissue samples are typically cut in micrometer thin sections, modern molecular detection
methods result in signals so densely packed that optical “slicing” by imaging at multiple focal planes becomes necessary to image all signals. Chromatic aberration, signal crosstalk and low signal to noise ratio further complicates the analysis of multiple sequences in parallel. Here a previous 2D analysis approach for image-based gene decoding was used to show how signal count as well as signal precision is increased when
analyzing the data in 3D instead. We corrected the extracted signal measurements for signal crosstalk, and improved the results of both 2D and 3D analysis. We applied our methodologies on a tissue sample imaged in six fluorescent channels during five cycles and seven focal planes, resulting in 210 images. Our methods are able to detect more than 5000 signals representing 140 different expressed genes analyzed and decoded in parallel.