Gradual labeling with fluorogenic probes for MINFLUX nanoscopy of densely packed structures

Abstract

Single-molecule localization in densely packed structures represents a formidable challenge in nanoscopy. Here we develop a gradual labeling method with fluorogenic probes for MINFLUX nanoscopy (GLF-MINFLUX), with which individual proteins in packed structures can be sequential illuminated and quantitatively localized at the nanoscale solely by adjusting probe concentration. With a 1.7-fold localization precision improvement and 2.2-fold faster acquisition, GLF-MINFLUX enables dual-channel nanoscale imaging of densely neuronal microtubules and microfilaments, and three-dimensional quantitative mapping of individual TOM20 proteins in mitochondrial clusters.