Multicolor 3D MINFLUX nanoscopy for biological imaging

Abstract

The resolution of conventional optical fluorescence far-field microscopes is limited by the diffraction of light. This implies that only features in a distance of about half of the wavelength can be discerned. In the last decades, the field of nanoscopy has evolved, theoretically promising molecular resolution by distinguishing close-by fluorescent emitters based on their molecular states that affect the molecules’ ability to fluoresce. Due to the limited number of photons that fluorescent molecules can emit before transitioning into a permanent dark state, the resolution of nanoscopy techniques remained limited to about 10–20 nm. The MINFLUX localization approach combines elements of different nanoscopy techniques to achieve true molecular resolution. By probing the position of individually emitting molecules with a targeted minimum of excitation light, the emitted photons are rendered more informative while leaving the photon budget untouched. Compared to a standard camera-based localization scheme, fewer photons are thus required to deduce the position of the molecule with a certain precision. At the inception of this work, MINFLUX delivered an unprecedented localization precision of around 1 nm when imaging isolated or cellular structures in two dimensions. Estimating the position of the molecule along the optical axis remained to be shown. Moreover, the MINFLUX implementation was limited to the acquisition of a single molecular species, preventing the study of intermolecular distances within biological objects. In this work, I present a MINFLUX nanoscopy approach offering isotropic nanometer precision in three dimensions. This is achieved by probing single molecules with a minimum of excitation light that is confined and targetable in all dimensions. I demonstrate high-fidelity multicolor MINFLUX imaging with molecular resolution in two and three dimensions. I further address the simultaneous tracking of more than one molecular species, which can potentially be applied for studying the dynamics of multi-component objects like protein assemblies. I demonstrate the applicability of 3D multicolor MINFLUX for biological imaging of proteins inside a cellular organelle. Together with an extensive analysis framework, I exploit the 3D isotropic nanometer localization precision as well as the multicolor imaging scheme for quantitatively studying the distribution of different proteins within the heterooligomeric MICOS protein complex in human mitochondria.2021-02-2