Nanodissection of Selected Viral Particles by Scanning Transmission Electron Microscopy/Focused Ion Beam for Genetic Identification.

This study presents the development of a new correlative workflow to bridge the gap between electron microscopy imaging and genetic analysis of viruses. The workflow enables the assignment of genetic information to a specific biological entity by harnessing the nanodissection capability of focused ion beam (FIB). This correlative workflow is based on scanning transmission electron microscopy (STEM) and FIB followed by a polymerase chain reaction (PCR). For this purpose, we studied the tomato brown rugose fruit virus (ToBRFV) and the adenovirus that have significant impacts on plant integrity and human health, respectively. STEM imaging was used for the identification and localization of virus particles on a transmission electron microscopy (TEM) grid followed by FIB milling of the desired region of interest. The final-milled product was subjected to genetic analysis by the PCR. The results prove that the FIB-milling process maintains the integrity of the genetic material as confirmed by the PCR. We demonstrate the identification of RNA and DNA viruses extracted from a few micrometers of an FIB-milled TEM grid. This workflow enables the genetic analysis of specifically imaged viral particles directly from heterogeneous clinical samples. In addition to viral diagnostics, the ability to isolate and to genetically identify specific submicrometer structures may prove valuable in additional fields, including subcellular organelle and granule research.