Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy

Zähringer et al. Light: Science & Applications (2023)12:70 https://doi.org/10.1038/s41377-023-01111-8 Official journal of the CIOMP 2047-7538 www.nature.com/lsa ARTICLE Open Access Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Jonas Zähringer 1 , Fiona Cole1, Johann Bohlen 1 , Florian Steiner 1,3 , Izabela Kamińska 1,2 and Philip Tinnefeld 1✉ Abstract 3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNAPAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNAPAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signalto-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds. Introduction 3D super-resolution with nanometer precision opens exciting new insights in nanostructures and biological systems by achieving molecular or even submolecular resolution. There is a multitude of techniques extending single-molecule localization microscopy (SMLM) to the third dimension, including PSF manipulation1,2, 4-Pi microscopy3, total internal reflection fluorescence (TIRF) microscopy4, repetitive optical selective exposure (ROSE-Z)5 or Supercritical Angle Localization Microscopy (SALM)6 and many more. However, in these techniques, the precision is mostly limited to the emission information, and hence the camera localization does not reach precisions of about the size of a fluorophore of Correspondence: Philip Tinnefeld () 1 Department of Chemistry and Center for NanoScience, Ludwig-MaximiliansUniversität München, Butenandtstr. 5-13 Haus E, 81377 München, Germany 2 Institute of Physical Chemistry Polish Academy of Sciences, Kasprzaka 44/52, 01-224, Warsaw, Poland Full list of author information is available at the end of the article 1–2 nm of all three dimensions. The coordinate-targeted approach of 3D stimulated emission depletion microscopy (STED)7 has similar limitations in precision. To this end MINFLUX nanoscopy8 and later MINSTED nanoscopy9 were introduced. By interrogating the emitter location with a series of targeted illuminations, localization precisions of <2 nm are reached with moderate photon budgets. It later was extended to 3D by superimposing vortex beams to generate a tophat10. However, the instrumental and engineering requirements increase with dimensionality and the photon budget is divided between the axial and lateral dimensions. Each photon only contributes to either the lateral or the axial localization depending on the kind of vortex mask of the respective illumination event. Alternative to optical approaches, the axial position of a fluorescent dye can be determined from near-field interactions with a modified coverslip. To this end, energy transfer between a dye and a metal- or graphenelayer is read out from fluorescence intensity or fluorescence lifetime and is converted to an axial information © The Author(s) 2023 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Zähringer et al. Light: Science & Applications (2023)12:70 Page 2 of 8 in approaches termed metal-induced energy transfer (MIET)11–13 or graphene energy transfer (GET)14–16. GET with graphene-on-glass coverslips has the advantage of high optical substrate transparency (>97%)17, lack of autofluorescence and steep d−4 distance dependence yielding the highest localization precision within its dynamic range14,18. In this work, we combine GET and pulsed-interleaved MINFLUX nanoscopy (pMINFLUX) with DNA-PAINT to enable nanometer precise 3D super-resolution imaging. pMINFLUX was introduced as simpler MINFLUX realization that additionally provides the fluorescence lifetime19. In combination with GET, axial position determination from the intensive property fluorescence lifetime is advantageous as it is intensity independent and does not require internal referencing. In the GETpMINFLUX combination, each photon is synergetically used for both, xy- as well as z-localization optimally exploiting the available information20. Using DNA origami nanopositioners, fluorescent molecules and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT), binding sites are placed precisely in 3D21. These nanopositioners are then used to evaluate the GETpMINFLUX DNA-PAINT combination for 3D localization and 3D super-resolution imaging at different distances to graphene14. To overcome the comparatively small field of view of pMINFLUX and the limited binding kinetics of DNA-PAINT, we also introduce local PAINT (L-PAINT) in which a DNA imager strand binds for 50 0 e Counts 300 150 0 z 0 25 Time [s] 4 8 12 16 Counts 100 20 z [nm] 10 20 30 40 Microtime [ns] 30 x 20 5 nm Counts y Fl. Int. [kHz] f 100 d z In GET-pMINFLUX nanoscopy, the xy position of a single fluorescent molecule placed on a graphene-on-glass coverslip using a DNA origami nanopositioner (Fig. 1a, top) is localized using pMINFLUX nanoscopy, while the axial position is determined by GET. To determine the 2D position of the dye it is excited by four spatially displaced and pulsed interleaved vortex beams19. By binning the fluorescent intensity trace (Fig. 1a, bottom), the number of photons corresponding to each of the four pulsed vortex beams is extracted via time-correlated singlephoto (...truncated)