Oral Presentation Australasian Cytometry Society 41st Annual Conference

Full scatter resolution of 80 nm polystyrene nanoparticles on a Beckman Coulter MoFlo AstriosEQ.   (24230)

Alexis Perez Gonzalez 1 , Tina Lee Luke 1 , Angela Hind 1 , Catherine Wai Chu Li 1
  1. University of Melbourne, Carlton, Melbourne, VICTORIA, Australia

Extracellular vesicle detection is a rapidly emerging application in Cytometry that challenges and unveils the field’s limitations in handling extremely dim signals. Whereas the contribution of fluorescence quantum efficiency and background to dim fluorescence detection, portray and resolution in Cytometry have been well established, scatter resolution of nanoparticles smaller than 200 nm brings an entirely new level of complexity into the Cytometer’s detectors playground. In the nano-realm, scatter detection difficulties derive from its non-linear size-dependency, the contribution of particle refraction index and laser wavelength, together with the overwhelmingly unfavorable imbalance between laser “noise” and nanoparticle’s feeble signals. This is the case for jet-in-air cell sorters such as Beckman Coulter MoFlo AstriosEQ, where particle interrogation collides with droplet formation as surface oscillations exponentially build up towards the jet’s break-off point. This work covers recent results while evaluating MoFlo AstriosEQ Forward and Side scatter detection limits for instruments located at two of the University of Melbourne Cytometry Platform nodes. Using nanoparticle beads mixes consisting of fluorescent and unstained polystyrene (PS), latex and silica beads of varying sizes (50-2,000 nm) and a procedure involving iterative fine-tuning of laser Beam Shaping Optics, nozzle and scatter assemblies, guided by resolution benchmarks in nanoparticle scatter-to-noise resolution indexes, the unique capacity of the Astrios to handle nanoparticle scatter in the context of minimal noise is revealed, with both instruments achieving full resolution of 80 and close to 110 nm PS nanoparticles from background at the 488 SSC and FSC detectors, respectively. We further evaluate the effect of a variety of factors on scatter resolution outcomes and unveil an unusual case of signal pulse entanglement between nanoparticles (Height) and noise (Width) at the very low detector’s signal end, with nanoparticles pulses seemingly lurking among the noise width ranks in a scatter signal height dependent manner.