Novel 3D super-resolution nanomicroscope

Recently, the team of Professor Philip Tinnefeld from the Department of Chemistry and Center for Nanoscience at the University of Munich, Germany, proposed to achieve 3D super-resolution nanomicroscopy by combining the 2D localization of pMINFLUX with axial information from graphene energy transfer (GET) and single-molecule switching by DNA-PAINT. He was published in the top international academic journal Light: Science & Applications under the title “Combining pMINFLUX, Graphene Energy Transfer and DNA-PAINT for Nanometer Precise 3D Super-Resolution Microscopy”.

Research background

3D super-resolution technology can achieve resolution at the molecular and even submolecular level, providing a completely new perspective on nanostructures and biological systems. Single-molecule localization microscopy (SMLM) has been extended into three dimensions, including PSF manipulation, 4-Pi microscopy, total internal reflection fluorescence microscopy (TIRF), repetitive optically selective exposure (ROSE-Z), or supercritical angular localization microscopy (SALM). However, in these techniques, camera positioning cannot achieve three-dimensional 1-2 nm fluorescent molecular accuracy. The coordinate localization methods of 3D stimulated emission loss (STED) microscopes have similar accuracy limitations. To this end, the researchers introduced the MINFLUX nanomicroscope and later the MINSTED nanomicroscope. By querying the emitter position using a series of targeted illuminations, it is possible to achieve an accuracy of less than 2 nm at medium photons, which can be extended to three dimensions by superimposing vortex beams to generate a “topological cap”. However, as the dimensions increase, so does the instrumentation and engineering, with photons distributed between axial and transverse dimensions. Each photon responds only to lateral or axial positioning.

In addition to optical methods, the axial position of the fluorescent dye can be determined by near-field interaction with the coverslip. For this purpose, the energy transfer between the dye and the metal or graphene layer is read from the fluorescence intensity or fluorescence lifetime and converted into axial information, called metal-induced energy transfer (MIET) or graphene energy transfer (GET) methods. The GET of graphene coverslips on a glass substrate has the advantages of high optical substrate transparency (>97%), no autofluorescence, and distance dependence on the order of d-4, which can achieve the highest positioning accuracy in its dynamic range.

Innovative research

3D nanoscale super-resolution microscopy is key to complementing fluorescence imaging and superstructure techniques. Professor Philip Tinnefeld’s team achieved three-dimensional super-resolution by combining the two-dimensional localization of pMINFLUX with axial information from graphene energy transfer (GET) and single-molecule switching from DNA-PAINT. pMINFLUX and GET represent a special synergistic combination suitable for near-surface super-resolution imaging, such as cell adhesion and membrane complexes, as information from each photon is used for two-dimensional and axial localization information. In addition, the researchers introduced local PAINT (L-PAINT), in which the DNA-PAINT imaging strand is equipped with an additional sequence for locally concentrated imaging. The combination of pMINFLUX, GET and DNA-PAINT represents a particularly efficient and versatile method for achieving three-dimensional super-resolution imaging with nanoscale precision, enabling the direct observation and analysis of complex structures and biological systems to unprecedented levels of detail, in addition to L-PAINT’s ability to image local aggregation.

Figure 1 Combining pMINFLUX with graphene energy transfer (GET) for precise 3D positioning. (a) Above: Schematic diagram of DNA structure where individual dyes are located at a height of 16 nm above graphene. Below: Trajectories of total fluorescence intensity of a single dye molecule in a single DNA origami structure. (b) Fluorescence attenuation signals in each of the four pulse-staggered vortex beams focused on samples arranged in a triangular pattern. The star represents the xy position of the dye molecule. (c) xy positioning histogram. (d) Fluorescence lifetime distribution. (e) Calculate the distance distribution of graphene z from the fluorescence lifetime of (d). (f) 3D localization of the full fluorescence intensity trajectory using the 2D information of pMINFLUX and the z-distance from the fluorescence lifetime. Individual positioning is shown in black, and the corresponding projections are shown on the side, with x, y at 1 nm and z at 0.2 nm.

The researchers showed that all three-dimensional positioning accuracy is less than 2 nm, and axial accuracy is below 0.3 nm. In 3D DNA-PAINT measurements, structural features (i.e., individual butt strands at a distance of 3 nm) are resolved directly on the DNA origami structure. These technologies are important for many fields, from basic biology and biophysics to materials science and nanotechnology, and are likely to open up many opportunities for scientific discovery and technological innovation in the coming years.

Figure 2 Performance of the combination of p-MINFLUX and GET. (a) Analysis of the distribution of distance z from graphene for exemplary molecules with different dye positions in the range of 12 to 30 nm with a fixed number of photons N=2000. (b) Exemplary 3D localization plot of a single stationary dye used to assess accuracy at a distance of 16 nm from graphene z, with varying numbers of photons N. (c) The positioning accuracy of x, y, and z is a function of the number of photons N of fixed dye molecules placed at different heights. The gray stripes indicate the x and y accuracy of MINFLUX in the corresponding experiment, and the lines represent the theoretical lower limit of accuracy.

Figure 3 Super-resolution DNA-PAINT imaging with pMINFLUX and GET. (a) Schematic diagram of the imaging structure with a protruding docking site. (b) 3D positioning of DNA-PAINT trajectories with corresponding projections on the sides. The division along the x- and y-axes is 1.5 nm, and the division along the z-axis is 0.5 nm. (c) Histogram of the distance of the selected x, y located to graphene z. (d) Histogram of x,y positioning relative to the average molecular position.

Figure 4 L-PAINT imaging using pMINFLUX and GET. (A schematic diagram of the L-PAINT imager harness.) (b) A DNA structure with three prominent strands. (c) Reduced DNA structure on graphene. (d) Fluorescence intensity trace with a red line at 2 sec. (e) 3D localization of L-PANT traces after 2 sec. (f) 3D positioning of the 30-sec L-PANT trajectory with corresponding projection on the side. (g) 2D histogram projected by xz, with a division of 1 nm and a positioning time division ranging from 15 to 50 ms. (h) Distance histogram to each average binding position on the x-axis. (i) Distance histogram to each average binding position on the y-axis. (j) Histogram of distance to graphene z.

The research was published in Light: Science & Applications under the title “Combining pMINFLUX, Graphene Energy Transfer and DNA-PAINT for Nanometer Precise 3D Super-Resolution Microscopy”. Jonas Zähringer is the first author and Philip Tinnefeld is the corresponding author. (Source: LightScience Applications WeChat public account)

Want to thesis information:https://www.nature.com/articles/s41377‍-023-0‍1111-8

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