image: Figure | 3D stabilization system for sub-10 nm microscopy. A, Scheme of the optical design, using gold nanoparticles for lateral drift correction and a reflected beam for axial stabilization. B, Sub-nm precision in the three dimensions maintained over 130 minutes of continuous operation. C, Comparison of p-MINFLUX imaging of a 10 nm separated DNA origami structure with and without stabilization.
Credit: Lucía F. Lopez et al.
Fluorescence nanoscopy has transformed our ability to visualize and discover biological structures. In recent years, ultraprecise single-molecule localization techniques such as MINFLUX and RASTMIN enabled visualization of nanostructures with molecular resolution at room temperature and in liquid environments, breaking new ground for structural and dynamical studies of both biological and artificial nanosystems. However, the widespread implementation of these techniques has been hindered by their significant technical and instrumental complexity. A critical challenge is the requirement for precise compensation of sample drift to achieve the highest possible precision during the relatively long measurement times. Now, researchers from Argentina, Germany, Poland, and Switzerland present an open-source, versatile solution in Light: Science & Applications: a stabilization system that achieves sub-nanometer precision for hours using simple optics and adaptable software.
The new stabilization system, described in Light: Science & Applications, combines a modular setup that can be added as a standalone module to an optical microscope with an open-source software called Takyaq. The system allows for active 3D stabilization, with the flexibility to independently correct for focus or lateral drift. The optical setup is built with standard components, and the software is designed for easy adaptation to a wide range of hardware. To demonstrate the system’s performance and adaptability, the team tested the stabilization system in two different setups, one used for pulsed interleaved MINFLUX (p-MINFLUX) and the other one for RASTMIN. In both instruments, they achieved sample drift stabilization below 1 nm for more than an hour. Specifically, the system achieved a remarkable stability of just 0.77 nm in lateral (XY) positioning and 0.76 nm in axial (Z) positioning, enabling the researchers to conduct long-duration single-molecule localization experiments with controlled drift-induced errors.
The authors note that the reported values were limited by the resolution of the piezo stage; therefore, improved stabilization could be achieved by using higher-end piezo stages, something that is easy to do thanks to the system’s adaptability. The software is hardware-agnostic, meaning it works with a wide range of cameras and stages, and it includes a user-friendly interface for real-time monitoring and adjustments. This flexibility lowers the complexity barrier for labs looking to implement ultra-high-resolution imaging. The team has made the software (github.com/Stefani-Lab/takyaq) and design files freely available, encouraging further development and collaboration.
“We consider that our system has the potential to be rapidly adopted by numerous research groups, substantially expanding the application field of single-molecule localization with true nanometer precision.”
Journal
Light Science & Applications
Article Title
Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy