Hidden Secrets Behind Dots: Improved Phytoplankton Taxonomic Resolution Using High‐Throughput Imaging Flow Cytometry

Phytoplankton are aquatic, microscopically small primary producers, accounting for almost half of the worldwide carbon fixation. As early indicators of environmental change, they play a crucial role in water quality management. Human activities like climate change, eutrophication, or international shipping traffic strongly impact diversity of these organisms. Phytoplankton monitoring is a crucial step in the recognition of changes in community composition. The common standard for monitoring programs is manual microscopic counting, which strongly limits sample number and sampling frequency. In contrast, high-throughput technologies like standard flow cytometry (FCM) are restricted to a low taxonomic resolution, which makes them unsuitable for the identification of indicator species. Imaging flow cytometers (IFC) could overcome these limitations as they combine microscopy and high-throughput analysis. In comparison to single fluorescence values, image information not only allows for a wide variety of possibilities to characterize different species as well as immediate and fast measurements but also provides an archivable data output. Taxonomic resolution of IFC (ImageStream X Mk II) was proven comparable to standard FCM (FACSAria II) by the help of numerical evaluations. This is demonstrated on different levels of taxonomic differentiation of laboratory grown cultures in this study. Phytoplankton species discrimination by an imaging flow cytometer could be useful as supportive tool to make machine-learning classifications more robust, reliable, and flexible. Furthermore, this study provides examples, demonstrating the possibility of discrimination between species with similar fluorescence properties, strains, and even subpopulations. In contrast to standard FCM, each cell is not only represented as a dot in a cytogram but is also linked to microscopic brightfield and the author presents a new way to visualize this as image-based cytograms. The source code is supplied and could be useful for all kind of IFC data in general. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.