A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles

Abstract

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

“…This issue, as well as other related questions, will be addressed in future studies. In this scenario, in a recent paper, the CytoFLEX was reported to detect EVs as small as 60 nm, triggering with VSSC and immunophenotyping [ 8 ]. While it is possible that the surface antigen density on the EVs used in that study was much higher than that of the EVs we tested here, it should be noted that the swarming issue was not addressed, so that it cannot be excluded that multiple small sized EVs coincidentally traversing the laser spot may have contributed to the detected fluorescence signal.…”

“…In the present study, we explored the feasibility to identify small/medium sized EVs (size range 70 to 300 nm) by a conventional flow cytometer, equipped with blue and violet laser excitation sources, designed to be used in clinical setting for a wide range of applications (CytoFLEX S, Beckman Coulter, Milano, Italy) [ 8 ].…”

Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles

“…The absolute purity of AF700 was determined to be 0.9556 g/g (gram of AF700 with counter ion per gram of AF700 powder) and 0.7124(60) g/g (gram of AF700 without counter ion per gram of AF700 powder). Applying the latter purity value to the concentration gives a value of 20.15 (17) mg AF700/kg DMSO solution (2.044(17) × 10 −5 mol/kg). Assuming a DMSO density of 1.0984 10 The purity by mass of each dye was determined with and without counter ion, because the stoichiometry found by qNMR for each dye was not an integer for the counter ion.…”

“…In order to be detected by scattering, particles also need to have a refractive index that is different from the flow fluid. Presently, general purpose flow cytometers that are commercially available have a size limit of about 80 nm [17]. Nano and quantum flow cytometers [25,29] are being developed to decrease this limit to 30 nm or smaller and typical sample volumes and number concentrations have not yet been established for these instruments.…”

“…Nano and quantum flow cytometers [25,29] are being developed to decrease this limit to 30 nm or smaller and typical sample volumes and number concentrations have not yet been established for these instruments. need standard size limit dilution error [24] Next Gen RPS 60 10 mL 10 6 to 10 10 need standard [18,19] FCM 80 100 mL 10 5 to 10 7 need standard [17] Quantum The virus counter is a specialized flow cytometer, designed to detect intact virus particles using a hydrodynamically focused nanostream and two fluorescence channels, one for nucleic acid detection and the other for capsid protein detection. No internal calibrant bead is required because the detected sample volume is determined by the instrument in real time.…”