Identification of RNA-containing virus particles using a triple correlative morphological and microscopic approach

Viral characterization based on different characteristics such as size or morphology is an important step towards a quick identification of virus infection. In this contribution, the identification and size determination of an isolated RNA virus with an atomic force microscope (AFM) in combination with conventional fluorescence microscopy is presented. Despite all the advantages of AFM for imaging individual viruses, discrimination by chemical composition and reliable differentiation of morphologically similar structures at the single particle level is hardly possible with the current state of the art. An effective method for identifying single virus particles based on their chemical structure is specific labeling and subsequent conventional fluorescence microscopic investigation. However, this method lacks morphologic information, and the resolution is generally above the virus size, impeding size determination. As a result, labeled hollow particles or viral fragments cannot be distinguished from intact particles of a similar size. In our experiments, the goal was to characterize and investigate the morphology, particularly the height of a model virus, here SARS-CoV-2 as an example, and unambiguously differentiate it from other sample components such as debris. The first step was topography mapping of inactivated virus samples using AFM. In the second step, the RNA-containing core and the spike proteins on the virus surface were labeled, and conventional fluorescence images were correlated with the topographic height images of the same particles, providing an accurate information about the actual stages of the viruses. As a main achievement, the experiments allow identification of individual intact SARS-CoV-2 particles and a distinction between viral fragments and potential false staining. The height range of virus particles under our specific inactivation conditions with paraformaldehyde treatment could be determined to be 60-110 nm. Following our results, pre-selection and distinction of viral particles from other sample components will be feasible by morphological characteristics alone, with a precision comparable to staining methods, prior to further specific identification. In general, our correlative approach combining information from scanning probe and conventional fluorescence microscopy can be applied to other virus strains as well, taken that virus-specific antibodies are known.