Trypanosomes and related parasites such as Leishmania are unicellular parasites with a precise internal structure. This makes light microscopy a powerful tool for interrogating their biology-whether considering advance techniques for visualizing the precise localization of proteins within the cell or simply measuring parasite cell shape. Methods to partially or fully automate analysis and interpretation are extremely powerful and provide easier access to microscope images as a source of quantitative data. This chapter provides an introduction to these methods using ImageJ/FIJI, free and open source software for scientific image analysis. It provides an overview of how ImageJ handles images and introduces the ImageJ macro/scripting language for automated images, starting at a basic level and assuming no previous programming/scripting experience. It then outlines three methods using ImageJ for automated analysis of trypanosome micrographs: Semiautomated cropping and setting image contrast for presentation, automated analysis of cell properties from a light micrograph field of view, and example semiautomated tools for quantitative analysis of protein localization. These are not presented as strict methods, but are instead described in detail with the intention of furnishing the reader with the ability to "hack" the scripts for their own needs or write their own scripts for partially and fully automated quantitation of trypanosomes from light micrographs. Most of the methods described here are transferrable to other types of microscope image and other cell types.
Methods in molecular biology (Clifton, N.J.)
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Nuffield Department of Medicine, The Peter Medawar Building for Pathogen Research University of Oxford, Oxford, UK. firstname.lastname@example.org.