Detergent lysis controls are critical for single extracellular vesicle phenotyping in SP-IRIS platforms

Abstract

Bulk analytical methods are extensively used to identify molecular cargo in extracellular vesicles (EVs), but they cannot distinguish nanovesicles from co-isolated non-vesicular particles or resolve discrete EV subpopulations. Single-particle interferometric reflectance imaging coupled with immunofluorescence (SP-IRIS/IF) has emerged as a platform to simultaneously resolve particle size, count, and cargo at single-EV resolution. However, differentiating authentic EVs from non-vesicular signals requires rigorous methodological controls that remain widely underreported in standard workflows. In this study, we analyzed human neural secretomes and serum EVs across different SP-IRIS/IF setups to evaluate their analytical specificity. By using detergent-mediated lysis controls, we found a critical technical limitation in standard single-particle analysis: the persistence of both interferometric and fluorescent signals after detergent treatment in a subset of presumed single-EV events that were non-vesicular artifacts. These findings indicate that lacking appropriate controls on single-EV platforms risks data misinterpretation, underscoring the indispensable implementation of detergent-mediated lysis for single-particle analysis in SP-IRIS/IF platforms.