Flow cytometric detection and sorting of EVs: analysis and characterization of background noise sources that may impact EV fluorescence or scatter detection. (24046)
EV detection via flow cytometry in fluorescence or scatter reaches the limits of current flow cytometry. Measurements of Q and B using an LED pulser indicates scatter and detection limits on the flow cytometer and is useful in understanding cytometer capabilities. However, understanding how electronic and optical noise sources may individually contribute to the system instrument noise pinpoints specific areas of instrument improvement for better EV detection. With high resolution electronics at and below the PMT dynamic range for the system, the noise background may be fully visualized, characterized and reduced. This poster describes both optical and electronic noise sources that may impact EV detection, characterization and sorting.
Methods: A MoFlo Astrios EQ (BEC) with a quantiFlash LED pulser (APE) installed were used to study noise sources such as: electronic (ADC, Preamp and PMT) and optical (laser, stream, nozzle tip distance, autofluorescence, nozzle tip sizes 25-100 um, and drop drive frequency and amplitude).
Optical and electrical noise determined the noise floor. The fluidic stream added 5-10% optical noise in fluorescent channels. Larger nozzle tip sizes reduced drop drive noise, whereas smaller nozzle tips reduced sort volume and optical noise but require higher pressure. About 5-10% of the optical noise is due to laser cross-talk.
Characterization and analysis of the sorter noise background provided insight into instrument EV characterization capabilities. Improvements to drop drive amplitude and frequency, laser selection and location and nozzle tip will reduce the noise floor. Optimal EV detection, isolation and characterization depends on measuring not only the Q value, but the B and true noise floor of the flow cytometer.
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