Computational protocol: Using Aiptasia as a Model to Study Metabolic Interactions in Cnidarian Symbiodinium Symbioses

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Protocol publication

[…] Gold-coated sections were imaged with the NanoSIMS 50 ion probe at the Center for Microscopy, Characterisation and Analysis at the University of Western Australia. Surfaces of samples were bombarded with a 16 keV primary Cs+ beam focused to a spot size of about 100 nm, with a current of ~2 pA. Secondary molecular ions 12C12C−, 12C13C−, 12C14N-, and 12C15N− were simultaneously collected in electron multipliers at a mass resolution (M/ΔM) of about 8,000, enough to resolve the 12C13C− from the 12C21H− peak and the 13C14N− and 12C15N− peaks from one another. Charge compensation was not necessary. Five images of different areas within the gastrodermis of the tentacle (25–45 μm raster with 256 × 256 pixels) were recorded for all targeted secondary molecular ions by rastering the primary beam across the sample with a dwell-time of 10–20 ms per pixel. After drift correction, the 13C/12C or 15N/14N maps were expressed as a hue-saturation-intensity image (HSI), where the color scale represents the isotope ratio. Image processing was performed using the ImageJ plugin OpenMIMS (National Resource for Imaging Mass Spectrometry, of the isotope labels was quantified for 20 Regions of Interest (ROIs) (circles of 2–10 μm) per category (symbiont cells, gastrodermal host tissue and gastrodermal vesicles) for each host–symbiont combination, and expressed using δ13C and δ15N notation. Gastrodermal host tissue was quantified in the form of ROIs placed adjacent to symbiont cells as clear cell boundaries were not always identifiable.Unlabeled Aiptasia served as unlabeled controls. δ13C and δ15N enrichment (expressed in %0) was quantified as follows:where N is the 15N/14N ratio of sample or unlabeled control and C is the 13C/12C ratio (measured as 12C13C−/12C12C− ions) of sample or unlabeled control, respectively. In this context, it is important to note that carbon and nitrogen incorporation at the cellular level was likely underestimated in our study as sample preparation for NanoSIMS may result in partial extraction of biomolecules. […]

Pipeline specifications

Software tools ImageJ, OpenMIMS
Applications Mass spectrometry imaging, Microscopic phenotype analysis
Diseases Disease Models, Animal
Chemicals Carbon, Nitrogen