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How long can I store my slides for?
Answer Slides stained with metal-labelled antibodies will remain stable for years at room temperature if stored correctly. Slides should be stored in a dry, dust-free environment. Cardboard boxes should be avoided. The best choice of container would be a plastic slide box.
How long will my samples take to run?
Answer Sample running time is mostly dependent on how many regions of interest (ROIs) are required for each section and how large each ROI is. It will take approximately 3 hours to ablate a 1mm2 ROI. Set up for ablation also takes some time. This can vary depending on factors such as whether co-localisation of images is required. Set-up time is also dependent on how long panoramas take to generate. If possible, we recommend cutting 2-3 sections per slide if space allows. This reduces slide set up time as it takes time to change between slides.
Will I need to compensate my data?
Answer Spill-over with imaging mass cytometry is minimal and is affected even less than suspension CyTOF. However, spill-over can still happen. In most cases this can be minimised with careful panel design and titration of antibodies. However, it is still possible to compensate imaging mass cytometry data using similar methods to those used for suspension CyTOF.
How long does the staining take?
Answer Typically, staining is done over two days with antibody staining at 4°C overnight. The first day of staining takes approximately 4.5 hours and the second day takes approximately 1.5 hours. These timings are based on protocols recommended by Fluidigm and may vary if alterations to the protocol are made.
Does Hyperion have a microscope so I can see my sample?
Answer The Hyperion is equipped with a camera which will produce a 20x magnification image of your slide. This camera can be used to create an image of a particular area of your slide – this is your ‘panorama’ from which you can choose specific areas to ablate. However, no other magnifications are available and the live slide image only covers a small area of the slide at a time. If a larger, more detailed view of your sample is needed to choose regions for ablation the we recommend co-localisation of your slide with a slide-scanned image from a serial section, or an image of your section taken on another microscope.
Do the files need processing?
Answer Hyperion data is exported as an MCD file which can be opened directly in certain programmes, such as MCD viewer without processing. For analysis in other programmes, such as CellProfiler, files should be in TIFF format. MCD viewer can be used to process your images into TIFF format, as well as other formats, so that they can be imported into other programmes for downstream analysis.
What software do I use for data analysis?
Answer Different software packages are available for the analysis of imaging mass cytometry data. Most commonly, Ilastik and CellProfiler are used for pixel classification and generation of cell segmentation masks. After generation of a cell segmentation mask to convert the image pixels into single cell events, data can then be analysed using HistoCAT or HistoCAT++, which were specifically designed for analysis of imaging mass cytometry data. For more information, see the ‘Hyperion data analysis’ section.