Single-Exosome Counting and 3D, Subdiffraction Limit Localization Using Dynamic Plasmonic Nanoaperture Label-Free Imaging

Abstract

Blood-circulating exosomes as a disease biomarker have great potential in clinical applications as they contain molecular information about their parental cells. However, label-free characterization of exosomes is challenging due to their small size. Without labeling, exosomes are virtually indistinguishable from other entities of similar size. Over recent years, several techniques have been developed to overcome the existing challenges. This article demonstrates a new label-free approach based on dynamic PlAsmonic NanO-apeRture lAbel-free iMAging (D-PANORAMA), a bright-field technique implemented on arrayed gold nanodisks on invisible substrates (AGNIS). PANORAMA provides high surface sensitivity and has been shown to count single 25 nm polystyrene beads previously. Herein, it is shown that using the dynamic imaging mode, D-PANORAMA can yield 3D, subdiffraction limited localization of individual 25 nm beads. Furthermore, D-PANORAMA's capability to size, count, and localize the 3D, subdiffraction limited position of individual exosomes is demonstrated as they bind to the AGNIS surface. The importance of both the in-plane and out-of-plane localization, which exploit the synergy of 2D imaging and the intensity contrast, is emphasized.