Authors

Roman Schmidt,corresponding author1 Tobias Weihs,1 Christian A. Wurm,1,2 Isabelle Jansen,1 Jasmin Rehman,2 Steffen J. Sahl,3 and Stefan W. Hellcorresponding author3,4

Affiliations

1Abberior Instruments GmbH, Göttingen, Germany. 2 Abberior GmbH, Göttingen, Germany. 3 Department of NanoBiophotonics, Max Planck Institute for
Biophysical Chemistry, Göttingen, Germany. 4Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany.

Topic

Biophotonics, Neuroscience

Abstract

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.

DOI: https://dx.doi.org/10.1038%2Fs41467-021-21652-z

Extract

The optical setup (Fig. 1a, Supplementary Figs. S1, S4, S7, and Supplementary Notes 1 and 2) is based on a common fluorescence microscope (IX83, Olympus) that provides routinely needed features such as compatibility with standard stages (U-780.DOS, Physik Instrumente), sample holders (P-545.SH4, Physik Instrumente), brightfield and epifluorescence illumination (CoolLED pE-4000, CoolLED) with filter sets and eyepieces for quickly checking large ROIs in the sample.

Product Associated Features

Full filter compatibility and 256 wavelength combinations from 16 LEDs (365 nm -770 nm) covers diverse experimental needs, while LEDs minimise photo-damamge when finding the field of view prior to analysis.

Product Type

pE-4000

Journal

Nature Communications

Year of Publication

2021

Country of Publication

Germany