High-Throughput Fingerprinting of Single Bio-Nanoparticles via Infrared Absorbance-Modulated Evanescent Scattering

Bio‐nanoparticles, including gene delivery vectors and extracellular vesicles, are essential in gene therapy, vaccine development, and disease diagnosis. Their intrinsic heterogeneity in size and molecular composition highlights the need for high-throughput and high-content single-particle analysis technologies. Here, we introduce infrared absorbance modulated evanescent scattering (IR-AMES) microscopy, an ultrasensitive bio-nanoparticle imaging platform. By harnessing an evanescent wave for orthogonal photothermal detection, IR-AMES achieves a 6.5‐fold probe field enhancement and doubles mid‐infrared illumination intensity using a gold-film-reflected substrate. Such capacity enables chemical imaging of 500‐nm nanoparticles at >18% modulation depth and 100 Hz frame rate, and fingerprinting of single adeno-associated virus (AAV) particles down to 26 nm in diameter with 187 nm lateral spatial resolution. IR-AMES is able to quantify the amount of RNA in single lentivirus vectors, differentiate DNA-containing and empty AAV vectors, and distinguish mRNA-loaded from empty lipid nanoparticles. IR-AMES imaging also reveals distinct chemical signatures of extracellular vesicles derived from human cells. Together, IR-AMES provides a versatile, non-destructive platform for analyzing other bio-nanoparticles spanning viruses, virus-like particles, and protein-based nanostructures.