Review of free software tools for image analysis of fluorescence cell micrographs

Summary An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs. We selected 15 free tools and divided them into stand‐alone, Matlab‐based, ImageJ‐based, free demo versions of commercial tools and data sharing tools. The review consists of two parts: First, we developed a criteria catalogue and rated the tools regarding structural requirements, functionality (flexibility, segmentation and image processing filters) and usability (documentation, data management, usability and visualization). Second, we performed an image processing case study with four representative fluorescence micrograph segmentation tasks with figure‐ground and cell separation. The tools display a wide range of functionality and usability. In the image processing case study, we were able to perform figure‐ground separation in all micrographs using mainly thresholding. Cell separation was not possible with most of the tools, because cell separation methods are provided only by a subset of the tools and are difficult to parametrize and to use. Most important is that the usability matches the functionality of a tool. To be usable, specialized tools with less functionality need to fulfill less usability criteria, whereas multipurpose tools need a well‐structured menu and intuitive graphical user interface. Lay Description Fluorescent micrographs depicting cell and cell structures are essential for many experiments in medicine and life sciences. For the assessment of such micrographs automatic and semi‐automatic image analysis tools, and more specifically, segmentation methods are essential. There are many free software tools available to assist scientists to assess fluorescence micrographs in various studies. Nevertheless, not every tool is suited for all users with little or no knowledge in image processing. Within this work we reviewed 15 free image processing tools for fluorescence micrographs evaluations and compared them in a twofold study: First, we developed a criteria catalogue and rated the tools regarding their usability, functionality and installation. Second, we performed an image processing case study and tried to solve typical image processing problems in fluorescen