Direct Extractionand Evaluation of Intraluminal VesiclesInside a Single Cell

Because endogenous extracellular vesicles are involved in important physiological functions, various techniques have been developed for their isolation and evaluation. However, methods for evaluating endogenous vesicles within cells are limited. This study presents a technique for the direct extraction and evaluation of intraluminal vesicles (ILVs). This technique combines scanning ion conductance microscopy, electrochemical syringes, and confocal microscopy to extract specific structures within a living cell, achieving high spatial resolution and accuracy at the femtoliter scale. This approach allowed the direct collection of CD63­(+) vesicles from HEK293 CD63-pHluorin-RFP cells and showed that their RNA expression profiles were different from those recovered from cytosol and extracellular vesicles isolated by ultracentrifuge. It also identified a subset specifically containing hsa-miR-145-5p and allowed for direct assessment of the local accumulation of miRNAs in cells. This technique is expected to become a powerful tool for evaluating the contents of ILVs within living cells.