ABSTRACT:

Super-resolution fluorescence microscopy has opened new possibilities for investigating biological structures at the nanoscale, with techniques such as single-molecule localization microscopy (SMLM) providing unprecedented spatial resolution. Yet the value of SMLM as a quantitative tool depends on three key aspects: how precisely molecules can be localized, how reliably imaging performance can be benchmarked, and how much information each molecule can report.

In this talk, I will present two complementary advances in super-resolution microscopy. First, I will discuss how combining SMLM with image scanning microscopy using a SPAD array detector enhances lateral resolution by up to a factor of two, while enabling fluorescence-lifetime-based multiplexing that avoids the chromatic registration errors associated with conventional spectral multiplexing.

Next, I will introduce bacteriophage T4 as a versatile three-dimensional biological nanoruler. Using DNA-PAINT combined with astigmatic imaging, we resolved its capsid and hollow tail in 3D, demonstrating how its well-defined architecture, preferential orientation, and multiple structural protein targets make it a promising reference for benchmarking super-resolution microscopy.

Finally, I will briefly outline future research directions aimed at extending SMLM beyond molecular localization towards probing local physicochemical environments at genetically defined sites within biological nanomachines.