Predictability in fluorescence spillover across flow cytometers, sample type, preparation methods and operators validates the design approach of pre-optimising fluorescence detectors.

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Using a flow cytometer to measure particle fluorescence has always been a relative, as opposed to an exact, science. Typically, flow cytometers rely on photomultipliers (PMTs) with a relatively limited dynamic range to detect and amplify fluorescence emissions by particles or cells. In order to capture the breadth of fluorescence encountered in either nature or the laboratory, the cytometrist must carefully set the voltage for each detector, in real time, or risk losing data. The methods most often employed for determining the "correct" voltage for each detector used in a given experiment are subjective and based on using negative and positive fluorescence controls. Positive fluorescence can only be defined in relation to appropriate amplification of a negatively fluorescent sample. There are many instances, however, where use of a negative sample to set voltage will lead to poor or erroneous results, such as when cells have high autofluorescence or have unexpectedly taken up fluorochrome. Additional examples include cases when there is no true negative control available, such as when analysing naturally fluorescent algae or phytoplankton, or when performing proliferation or membrane potential assays where all cells are pre-loaded with fluorochrome.

The design of the Accuri® C6 Flow Cytometer® System addresses the subjective art of setting voltage by standardising fluorescence detection on every instrument at manufacture. The C6 was electronically engineered to expand the effective dynamic range from the standard four or five decades to 6.2 decades utilising 24-bit digital signal processing and advanced noise reduction processes. During manufacture, laser and optical alignments are set and locked down. Finally, voltage settings for each detector are optimised using industry-standard beads (Spherotech Rainbow Fluorescent Particles). These beads fluoresce across a broad range of wavelengths and can be used to adjust voltage on every channel. Rough, and then fine, voltage adjustments are made which place the brightest bead peak in a specified channel for each detector, while maintaining good low-end resolution for dimmer peaks. The result is that each C6 Flow Cytometer is manufactured with standardized fluorescence performance. End users do not need to adjust voltage.