Stitching and registering highly multiplexed whole-slide images of tissues and tumors using ASHLAR

Bioinformatics. 2022 Sep 30;38(19):4613-4621. doi: 10.1093/bioinformatics/btac544.


Motivation: Stitching microscope images into a mosaic is an essential step in the analysis and visualization of large biological specimens, particularly human and animal tissues. Recent approaches to highly multiplexed imaging generate high-plex data from sequential rounds of lower-plex imaging. These multiplexed imaging methods promise to yield precise molecular single-cell data and information on cellular neighborhoods and tissue architecture. However, attaining mosaic images with single-cell accuracy requires robust image stitching and image registration capabilities that are not met by existing methods.

Results: We describe the development and testing of ASHLAR, a Python tool for coordinated stitching and registration of 103 or more individual multiplexed images to generate accurate whole-slide mosaics. ASHLAR reads image formats from most commercial microscopes and slide scanners, and we show that it performs better than existing open-source and commercial software. ASHLAR outputs standard OME-TIFF images that are ready for analysis by other open-source tools and recently developed image analysis pipelines.

Availability and implementation: ASHLAR is written in Python and is available under the MIT license at The newly published data underlying this article are available in Sage Synapse at; the availability of other previously published data re-analyzed in this article is described in Supplementary Table S4. An informational website with user guides and test data is available at

Supplementary information: Supplementary data are available at Bioinformatics online.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Data Collection
  • Humans
  • Image Processing, Computer-Assisted / methods
  • Microscopy / methods
  • Neoplasms* / diagnostic imaging
  • Software*