Fluorescence Microfluidic Resistive Pulse Sensing (F-MRPS, Spectradyne) was used to measure Vesi-Ref CD63 GFP (Vesiculab, UK) without modification and after staining with PE-anti-CD63. F-MRPS detects and measures the size and concentration of nanoparticles using MRPS, an electrical technique, while simultaneously measuring the fluorescence of each particle in up to 3 optical bands. Particle detection and fluorescent measurements are triggered using the MRPS signal, ensuring that each event represents a physical particle larger than the size limit of detection of the cartridge used (65 nm in this case). A one-time factory cross-calibration of the instrument to NIST-traceable MESF standard beads enables particle brightness to be reported by default in traceable units (ERF or MESF when appropriate).

The main figure shows particle concentration vs. size for all particles in the unstained sample (purple), all particles in the stained sample (blue), and subpopulations of the stained sample after gating GFP+ (green), CD63+ (yellow), and double-positive GFP+/CD63+ (red) populations. While the overall particle size distribution displays an approximate power-law dependence of concentration on size that extends to the lower size limit of detection for this cartridge, the fluorescent subpopulations each show the presence of a peak near 75 nm diameter. The 2D scatter plot (inset) shows the brightness of each particle in FITC and PE channels. Absolute concentration and relative abundance of particles are reported for each quadrant, the bounds of which are defined by the fluorescence limits of detection of each channel: 30 FITC ERF and 2.5 PE MESF.

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