The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method’s general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1–3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

Formylation of 2,6‐dichloro‐5‐R‐nicotinic acids at C‐4 followed by condensation with 3‐hydroxy‐N,N‐dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6‐dichloro‐5‐R‐nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with “small molecules” provided specific labeling (covalent and non‐covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two‐color STED microscopy with a 775 nm STED laser.

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are required. In addition, the fluorophores should preferentially emit in the red spectral range to reduce potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. Here we demonstrate two-color STED imaging of living cells using various pairs of dyes that fulfill all of the above requirements. To this end, we combine click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, enabling long-term STED imaging of different target structures in living specimens.