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

## TL;DR

This study combines atomic force microscopy and fluorescence imaging to accurately identify SARS-CoV-2 particles based on their size, shape, and molecular features.

## Contribution

A novel tripartite imaging strategy that integrates AFM and double-staining fluorescence for high-specificity RNA virus identification.

## Key findings

- SARS-CoV-2 particles were identified with a height range of 60–100 nm using AFM and fluorescence correlation.
- The method distinguishes intact SARS-CoV-2 from hollow particles, fragments, and artifacts.
- The approach is broadly applicable to other RNA viruses for high-specificity detection.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1186/s12967-025-07490-3.