Super‐Resolution Goes Viral: T4 Virus Particles as Versatile 3D‐Bio‐NanoRulers

Abstract In the burgeoning field of super‐resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the 3D domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of 3D subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub‐diffraction‐limit distances. In this context, the use of the non‐human‐infecting virus, bacteriophage T4 is introduced as an effective and straightforward bio‐ruler for 3D super‐resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA‐PAINT) along with the technique of astigmatic imaging, the icosahedral capsid of the bacteriophage T4, measuring 120 nm in length and 86 nm in width, and its hollow viral tail is uncovered. This level of detail in light microscopy represents a significant advancement in T4 imaging. A simple protocol for the production and preparation of samples is further outlined. Moreover, the extensive potential of bacteriophage T4 as a multifaceted 3D bio‐ruler, proposing its application as a novel benchmark for 3D super‐resolution imaging in biological studies is explored.