Buffer-Free MINFLUX Imaging via Nanobody Points Accumulation

Abstract

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, the recently developed super-resolution microscopy method, excels in acquiring high-fidelity images with single-nanometer resolution. The initial implementation of MINFLUX involves separating neighboring fluorophores by individually switching their emission on/off through a complex redox-active blinking buffer, a process found to be operationally cumbersome. While numerous MINFLUX imaging techniques have utilized immobilized probes for nanoscale fluorescence imaging, few have leveraged their binding kinetics. Our study demonstrates the efficacy of MINFLUX imaging facilitated by the accumulation of anti-GFP nanobody at GFP–protein fusions, without the need for additional substances. We explore and adapt nanobody-targeted MINFLUX imaging for diverse subcellular structures. Notably, our approach enables the visualization of the hollow structure of cellular microtubules for the first time in a MINFLUX imaging application. Simultaneously, we performed a comparative analysis of subcellular structure images obtained via STED microscopy. Our labeling technique offers a straightforward and adaptable method for achieving MINFLUX nanoscopy.