Here, University of Amsterdam researchers present a new method, named flow cytometry scatter ratio (Flow-SR), to relate the ambiguous light scattering signals of flow cytometry to the diameter and refractive index (RI) of single nanoparticles between 200-500 nm in diameter. Flow-SR enables label-free differentiation between EVs and LPs and improves data interpretation and comparison. Because Flow-SR is easy to implement, widely applicable, and more accurate and faster than existing techniques to size nanoparticles in suspension, Flow-SR has numerous applications in nanomedicine.
Validation of size and refractive index (RI)
determination of nanoparticles by the flow cytometry scatter ratio
Validation of size and refractive index (RI) determination of nanoparticles by the flow cytometry scatter ratio (Flow-SR) using a bead mixture containing (A) 125 nm, (B) 240 nm, (C) 315 nm, and (D) 380 nm polystyrene beads and (E) 255 nm, and (F) 391 nm silica beads, oil emulsions, Intralipid, and gold nanoparticles. (a) Scatter plot of side scatter versus forward scatter of the bead mixture. Due to the arbitrary units (a.u.), the diameter and RI of the beads are unknown. (b) Scatter plot of RI versus diameter of the bead mixture. The diameter is obtained from Flow-SR. The RI is derived from a lookup table of forward scatter versus diameter. Bead populations B-F are clearly differentiated by size and RI. Bead population A and background noise result in a horizontal and vertical asymptote, caused by the detection thresholds and limited resolution of the flow cytometer. Panel (a) and (b) share the same scale bar. (c) Size distributions of the bead mixture (solid lines) fitted by Gaussian distributions (dotted lines). The bin width is 10 nm. The vertical bars indicate the reference size as mean ± standard deviation. (d,e) RI distributions (lines) of the bead mixture and oil emulsions, Intralipid, and 200 nm gold nanoparticles. The vertical bars indicate the range of reported RIs from literature.