Nanoscale single-vesicle analysis: High-throughput approaches through AI-enhanced super-resolution image analysis.

The analysis of membrane vesicles at the nanoscale level is crucial for advancing the understanding of intercellular communication and its implications for health and disease. Despite their significance, the nanoscale analysis of vesicles at the single particle level faces challenges owing to their small size and the complexity of biological fluids. This new vesicle analysis tool leverages the single-molecule sensitivity of super-resolution microscopy (SRM) and the high-throughput analysis capability of deep-learning algorithms. By comparing classical clustering methods (k-means, DBSCAN, and SR-Tesseler) with deep-learning-based approaches (YOLO, DETR, Deformable DETR, and Faster R-CNN) for the analysis of super-resolution fluorescence images of exosomes, we identified the deep-learning algorithm, Deformable DETR, as the most effective. It showed superior accuracy and a reduced processing time for detecting individual vesicles from SRM images. Our findings demonstrate that image-based deep-learning-enhanced methods from SRM images significantly outperform traditional coordinate-based clustering techniques in identifying individual vesicles and resolving the challenges related to misidentification and computational demands. Moreover, the application of the combined Deformable DETR and ConvNeXt-S algorithms to differently labeled exosomes revealed its capability to differentiate between them, indicating its potential to dissect the heterogeneity of vesicle populations. This breakthrough in vesicle analysis suggests a paradigm shift towards the integration of AI into super-resolution imaging, which is promising for unlocking new frontiers in vesicle biology, disease diagnostics, and the development of vesicle-based therapeutics.