Atomic force microscopy-based topographical imaging of SARS-CoV-2 as part of a tripartite strategy for RNA virus characterization.

BACKGROUND

The pre-selection of virus particles based on size and morphology is a crucial step toward rapid and reliable virus identification. Pre-selecting virus particles based on size and morphology represents a critical step toward rapid and reliable identification, which is particulary important in clinical settings when novel virus variants emerge. Although conventional fluorescence imaging enables visualization of specific viral structures via labeling, it does not allow for reliable differentiation of structurally similar particles.

METHODS

In this study, we present a combined imaging approach that integrates atomic force microscopy (AFM) and double-staining fluorescence microscopy to identify SARS-CoV-2 as a model RNA-virus from other sample constituents.

RESULTS

Initially, topographical imaging via AFM enables high-resolution visualization of individual virus particles, providing detailed information about particle morphology and height. Subsequently, dual fluorescence labeling of the RNA-containing core and the spike protein-rich surface allows for specific identification of intact viral structures. Correlation of fluorescence signals with AFM-derived height maps offers a comprehensive view of the particles’ morphological and molecular characteristics.

CONCLUSION

This triple-correlation strategy enables the identification of intact SARS-CoV-2 particles and their clear distinction from similarly sized hollow particles, viral fragments, and staining artifacts. The height range of confirmed SARS-CoV-2 particles under the applied conditions was determined to be 60–100 nm. The presented correlative imaging approach is broadly applicable to other RNA viruses, offering a versatile tool for high-specificity virus detection.