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Background  A range of spatially resolved transcriptomic methods has recently emerged as a way to spatially characterize the molecular and cellular diversity of a tissue. As a consequence, an increasing number of computational techniques are developed to facilitate data analysis. There is also a need for versatile user friendly tools that can be used for a de novo exploration of datasets.    Results  Here we present MATLAB-based Analysis toolbox for in situ sequencing (ISS) expression maps ( Matisse ). We demonstrate  Matisse  by characterizing the 2-dimensional spatial expression of 119 genes profiled in a mouse coronal section, exploring different levels of complexity. Additionally, in a comprehensive analysis, we further analyzed expression maps from a second technology, osmFISH, targeting a similar mouse brain region.    Conclusion   Matisse  proves to be a valuable tool for initial exploration of in situ  sequencing  datasets .  The wide set of tools integrated allows for simple analysis, using the position of individual reads, up to more complex clustering and dimensional reduction approaches, taking cellular content into account. The toolbox can be used to analyze one or several samples at a time, even from different spatial technologies, and it includes different segmentation approaches that can be useful in the analysis of spatially resolved transcriptomic datasets.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12859-021-04302-5.

Matisse: a MATLAB-based analysis toolbox for in situ sequencing expression maps