Improved forward scatter detection of a flow cytometer for detection of extracellular vesicles

Abstract

Extracellular vesicles (EVs) are nanoparticles secreted from cells into bodily fluids. EVs are potential biomarkers for diseases such as thrombosis or cancer. However, the small size and low refractive index of EVs complicates their detection. A flow cytometer is suited for EV characterisation, but typically lacks scatter sensitivity on one or both scatter detectors for derivation of both particle size and refractive index. Here, we aim to improve the FACSCanto (Becton Dickinson) forward scatter detector for the detection of 100 nm EVs, which requires an improvement in SNR of 107-fold based on Mie theory. This was achieved through replacement of the 20 mW laser by an 200 mW laser, replacement of the photodiode detector with a photomultiplier tube and a confocalized optical geometry. Using a prototype optical setup, we obtained an improvement in SNR which was 1,11·104 – fold better than the standard design. However, the optics was suboptimal and far from diffraction-limited. Zemax simulations led to a nearly diffraction limited optical design which is expected to yield another 200-fold improvement. Taken together these changes will improve the SNR 2.2·106-fold and thus improve the detection limit of the FACSCanto to 130 nm EVs.