Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis
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Open Access
- 3 January 2012
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Biology
- Vol. 10 (1), 1
- https://doi.org/10.1186/1741-7007-10-1
Abstract
Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method. 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.Keywords
This publication has 46 references indexed in Scilit:
- The Kinetoplast Duplication Cycle in Trypanosoma brucei Is Orchestrated by Cytoskeleton-Mediated Cell MorphogenesisMolecular and Cellular Biology, 2011
- The cell cycle of Leishmania: morphogenetic events and their implications for parasite biologyMolecular Microbiology, 2010
- Image-Based High-Throughput Drug Screening Targeting the Intracellular Stage of Trypanosoma cruzi , the Agent of Chagas' DiseaseAntimicrobial Agents and Chemotherapy, 2010
- Automated characterization of cell shape changes during amoeboid motility by skeletonizationBMC Systems Biology, 2010
- Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imagingMolecular and Biochemical Parasitology, 2008
- Functional characterization of cohesin subunit SCC1 in Trypanosoma brucei and dissection of mutant phenotypes in two life cycle stagesMolecular Microbiology, 2008
- Functional genomics in Trypanosoma brucei: A collection of vectors for the expression of tagged proteins from endogenous and ectopic gene lociMolecular and Biochemical Parasitology, 2007
- Basal Body Positioning Is Controlled by Flagellum Formation in Trypanosoma bruceiPLOS ONE, 2007
- NeuronMetrics: Software for semi-automated processing of cultured neuron imagesBrain Research, 2007
- Chromosome-Wide Analysis of Gene Function by RNA Interference in the African TrypanosomeEukaryotic Cell, 2006