Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis

We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically.Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.Trypanosomatids are a family of single celled parasitic protozoa that includes several major human pathogens, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which are responsible for a range of tropical and subtropical diseases. All trypanosomatids share similar, precisely defined, architectures; a single flagellum that emerges from an invagination of the plasma membrane at its base (the flagellar pocket), the kinetoplast (mitochondrial DNA, which can make up a large proportion of total DNA, for example, 30%, for T. cruzi [1,2]) found within the single mitochondrion at the base of the flagellum, a nucleus, a