When analysing small particles, defined as particles smaller than 3.0µm, on the Accuri C6 certain items must be considered to ensure proper data acquisition. This note discusses some of the basic concepts needed to perform small particle acquisition and analysis properly.

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When analysing small particles, defined as particles smaller than 3um, on the Accuri C6 certain items must be considered to ensure proper data acquisition. This note discusses some of the basic concepts needed to perform small particle acquisition and analysis properly.

Thresholds

A threshold is the minimum requirement an event must pass to be recorded by the cytometer. The trigger channel is the critical parameter used to determine if an event should be recorded. When using the C6, setting the primary threshold also defines the trigger channel.

On the C6, the signal width is defined as threshold cross as the signal increases to threshold cross as the signal decreases (time of flight). On the C6, the default setting of a primary threshold on FSC-H of 80,000 indicates the C6 will only record an event if it has a FSC value of 80,000 regardless of any other parameter values. Using this setting, the 8-Peak and 6-Peak Validation Beads can easily be visualised on a FSC/SSC plot. All thresholds on the C6 are applied via the Height signal measurements. There is no impact on the data if the area parameter is viewed during data acquisition or analysis. However this will sometimes lead to a discrepancy in where the threshold falls when viewing Area data. The difference between where the data is thresholded in area vs. height will vary depending on the particles being analysed.

The reason for this difference is that the area data is being displayed after area scaling factors have been applied. The C6 uses fixed area scaling factors which do not normalise the area data values exactly to the height data values for all sample types.

Setting the threshold

When choosing the appropriate threshold value, debris and undesired events should be eliminated while not inadvertently eliminating relevant events. lf standard, or familiar, samples are being acquired, the threshold can be set based on experience. However, if this is the first time a particular experiment is performed, it may not be clear which events are the correct ones, so they must be identified. Once the particles have been identified, the threshold should be set low enough to ensure the entire population is captured. One way to do this would be to set the threshold to its lowest value (ten). However, setting the threshold very low on any parameter will result in the electronic noise for that channel becoming visible and interfering with acquisition and analysis.

The noise floor

On all flow cytometers, every channel has an inherent noise floor. Noise is comprised of low level signals that occur within the electronics and of stray light being collected by the optics. This noise appears as events that are collected even when there are no, or very few, "true" events in the sample. For example, a sample from a tube of 0.2 um, filtered water was acquired at the standard settings and only a small number of events were detected. These are most likely "true" particulate matter. As the threshold is lowered, more ancl more events are detected until those events dominate the plot. With the threshold set to 80,000, acquiring sample for 1 min resulted in only 80 events. However, the same sample re-acquired with a threshold of 1,000 resulted in >200,000 events.