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, researchers from Beckman Coulter Life Sciences assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. They found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). The researchers 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, they 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, the researchers 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. They 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.
Optical Innovations in the CytoFLEX
(A) Wavelength-division multiplexing is a method for parsing ranges of light wavelengths, adapted from fiber-optics technology used in the telecommunications industry. Input light from the fiber-optic cable is sequentially reflected by bandpass filters until the particular wavelength range encounters a permissive filter that allows the light to pass through to its associated APD. This design minimizes the loss of light, as occurs with dichroic mirrors. (B) The catadioptric flow-cell design maximizes light collection. Approximately 110° of side-scatter and fluorescent light is focused by a plano-concave mirror on the back of the flow cell. The light path is then shaped by a lens designed similar to a Schmidt corrector plate, which directs light originating from the different lasers to their respective fiber-optic pinholes, while also minimizing the cross-mixing of light.
Overall, the researchers 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.