Metrology of Individual Small Viruses

Viruses come in various shapes and sizes, and understanding their morphology is central to understanding their activity and function. The need for fast recognition and real‐time fingerprinting methods for pathogenic viruses is a critical bottleneck in implementing many diagnostic and therapeutic techniques. In this work, nanopore tomography (NT) is implemented for fast measurements of the characteristic dimensions of viruses and the optimal operating conditions are explored. Using a small filamentous bacteriophage as a model, it is demonstrated that NT can detect geometrical features in a few‐nanometer regime, with high throughput and accuracy, in aqueous conditions. The instrumental parameters are optimized to obtain virus diameter measurements that are robust to the uncertainties of the external parameters. Furthermore, NT is critically compared to various single‐particle imaging techniques, with a particular emphasis on emerging helium ion microscopy (HIM). It is shown that, with proper operating procedures, HIM can reach a nanometer‐scale resolution in viral metrology, while retaining a high throughput second only to NT. The high throughput of both techniques can foster sufficient statistics for a precise exploration of viral heterogeneity. Understanding virus morphology is crucial for insights into their activity. Nanopore tomography (NT) offers fast, accurate measurements of virus dimensions in aqueous solutions. Instrumental parameters are optimized to obtain robust and reliable measurements. Helium ion microscopy also shows nanometer‐scale resolution and high throughput, second only to NT, fostering precise exploration of viral heterogeneity.