Prevalence and practices of immunofluorescent cell image processing: a systematic review

We performed a systematic review that identified at least 9,000 scientific papers on PubMed that include immunofluorescent images of cells from the central nervous system (CNS). These CNS papers contain tens of thousands of immunofluorescent neural images supporting the findings of over 50,000 associated researchers. While many existing reviews discuss different aspects of immunofluorescent microscopy, such as image acquisition and staining protocols, few papers discuss immunofluorescent imaging from an image-processing perspective. We analyzed the literature to determine the image processing methods that were commonly published alongside the associated CNS cell, microscopy technique, and animal model, and highlight gaps in image processing documentation and reporting in the CNS research field.

We completed a comprehensive search of PubMed publications using Medical Subject Headings (MeSH) terms and other general search terms for CNS cells and common fluorescent microscopy techniques. Publications were found on PubMed using a combination of column description terms and row description terms. We manually tagged the comma-separated values file (CSV) metadata of each publication with the following categories: animal or cell model, quantified features, threshold techniques, segmentation techniques, and image processing software.

Of the almost 9,000 immunofluorescent imaging papers identified in our search, only 856 explicitly include image processing information. Moreover, hundreds of the 856 papers are missing thresholding, segmentation, and morphological feature details necessary for explainable, unbiased, and reproducible results. In our assessment of the literature, we visualized current image processing practices, compiled the image processing options from the top twelve software programs, and designed a road map to enhance image processing. We determined that thresholding and segmentation methods were often left out of publications and underreported or underutilized for quantifying CNS cell research.

Less than 10% of papers with immunofluorescent images include image processing in their methods. A few authors are implementing advanced methods in image analysis to quantify over 40 different CNS cell features, which can provide quantitative insights in CNS cell features that will advance CNS research. However, our review puts forward that image analysis methods will remain limited in rigor and reproducibility without more rigorous and detailed reporting of image processing methods.

Image processing is a critical part of CNS research that must be improved to increase scientific insight, explainability, reproducibility, and rigor.