Detection and enumeration of single nanometric particles: A confocal optical design for fluorescence flow cytometry

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Abstract
Nanometer-sized particles are increasingly important in fields ranging from technology development to environmental analysis. Traditionally, quantification of biological nanometric particles, such as viruses, has been challenging. Recent advances in optics and optical design have made single molecule detection possible in flowing systems. However, many commercially available flow cytometers are not optimized for routine analysis of ultrasmall biological particles. In this work, a confocal optical arrangement was incorporated into the design of a relatively simple flow cytometer optimized for rapid enumeration of fluorescent nanometric particles. The instrument was designated the “single nanometric particle enumerator” (SNaPE). Instrument parameters, such as data acquisition rates, flow rates, and minimum sampling time were examined and optimized for the SNaPE. The measured detection efficiency was ∼8%, which is consistent with the confocal geometry and equivalent to the detection efficiency reported in many single molecule studies. Signal calibration was achieved using fluorescent polystyrene spheres ranging from 26 nm to 10 μm in diameter. The SNaPE exhibited a limit of detection of ∼180 fluorescein isothiocyanate equivalents and was capable of particle enumeration over the tested concentration range of 106–109 particles/ml.