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Mass cytometry offers a few unique advantages over standard fluorescence based flow cytometry:
(Nearly) no compensation – an intrinsic issue with fluorescence based flow cytometry is the ‘spill’ of signal from individual florescent molecules into neighbouring detectors or generally across the detection system. This has to be accounted for using a correction method called ‘compensation’. As compensation is a subtractive method it can cause loss of sensitivity to some channels, especially important if signal is low due to low expression. There is (nearly) no compensation associated with mass cytometry due the collection of metal ions present on cells, which exhibit a unique atomic mass. However, some ‘spill’ can occur due to impurities in the metals or due to oxidation. This spill however is both minimal (<10% compared to >30% exhibited by some florescent molecules) and predictable. This in turn makes panel design relatively straightforward.
Increased parameters – with 35 isotypically unique metals now available as pre conjugated or available to conjugate to antibodies and probes mass cytometry offers a high degree of multidimensionality for single cell research.
No autofluorescence - as mass cytometry doesn’t collect fluorescence there is no issue with autofluorescence (AF)! This is not often a problem in standard flow cytometry as the range of detection allows us to use bright flurochromes which overcomes issues with AF. However some cell types, such as macrophages, are simply un-readable using fluorescence. Mass cytometry offers a real advantage in these situations.
Limited sample – single cocktail on all sample, controls (unlabelled, titrations)
Samples of cells are prepared with a protocol in the same manner as standard flow cytometry to generate a antibody labelled single cell suspension. Antibodies tagged with elemental isotopes are then introduced into the mass cytometer. Cells are vapourised into droplets containing single cells by being sprayed out of a nebuliser and ionised as they pass through an induction coupled plasma. These ion clouds, each one representing a single cell event are filtered to remove low mass ions, accelerated through a time of flight chamber and the ion content per cloud is measured. The signal for each channel from each cell is used to generate a listmode file which can be read by any flow cytometry data analysis package or uploaded to Cytobank for further analysis.
Helios system
We have the most recent mass cytometer produced by Fluidigm in the form of the Helios. The Helios offers a number of improvements over the previous two versions, the CyTOF and CyTOF 2. Article in The Scientist
Namely:
easy to use software user interface
real time data display
more automated QC and normalisation protocols
wider range of detection allowing for potentially 135 channels
improved sample delivery system for consistant sample introduction
more efficient cell transition (less cell loss)
barcoding
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