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Super-Resolution Microscopy for 3D Imaging

Three-dimensional (3D) super-resolution microscopy is essential to study the minute structural details of cells and analyze molecular dynamics. Recently, researchers at Ludwig-Maximilians-Universität München, Germany, have developed a super-resolution microscopy method that can rapidly differentiate molecular structures in 3D. This study is available in Light Science and Applications.

Super-Resolution Microscopy for 3D Imaging

a Top: Schematic of a DNA origami structure with a single dye positioned at a height of 16 nm above a graphene-on-glass coverslip. Bottom: Fluorescence intensity trace of the total fluorescence intensity of a single dye molecule in a single DNA origami structure. b Fluorescence decays for each of the four pulsed interleaved vortex-shaped beams which are focused on the sample arranged in a triangular pattern with the fourth beam placed at the center of the triangular structure. The star indicates the xy position of the dye molecule. c xy localization histogram of time bins. d Distribution of fluorescence lifetimes obtained from the time bins. e Distribution of the distances to graphene z calculated from the fluorescence lifetimes of d). f 3D localizations of the full fluorescence intensity trace using the 2D information of pMINFLUX and the z distances from the fluorescence lifetimes. The individual localizations are shown in black and on the sides the corresponding projections with a binning of 1 nm for xy and 0.2 nm for z © Zähringer, J. et al. (2023)

Studying Objects at Nanometric Dimensions

3D super-resolution microscopy provides a better understanding of nanostructures and biological features at molecular or submolecular precision. Several microscopic techniques advanced single-molecule localization microscopy (SMLM) in 3D, including total internal reflection fluorescence (TIRF) microscopy, 4-Pi microscopy, Supercritical Angle Localization Microscopy (SALM), and repetitive optical selective exposure (ROSE-Z). Nevertheless, these techniques are mostly limited to emission information and lack 3D precision in imaging.

The shortcoming of the above-mentioned microscopy was overcome by MINFLUX nanoscopy followed by MINSTED nanoscopy, which enabled localization precisions of less than 2 nanometers (nm) with adequate photon budgets. This technique was later extended to 3D by introducing superimposed vortex beams to produce top-hat.

Nevertheless, this technique is complex, requiring advanced equipment and engineering. In addition, the photon budget was divided into lateral and axial dimensions, where each photon was allocated to either one of the two dimensions. This allocation is dependent on the vortex type based on the illumination event.

Alternatively, a fluorescent dye can be used to determine the axial position in near-field microscopy, utilizing a modified coverslip. In some recent research, the energy emission and transfer from a dye to a metal/graphene layer were determined based on the intensity and shelf-life of the fluorescence. This information is transformed into axial information using various techniques, such as metal-induced energy transfer (MIET) and graphene energy transfer (GET).

Two main advantages of GET with specialized glass coverslips containing graphene are high optical substrate transparency and lack of autofluorescence. This technique also provides superior localization precision within its dynamic range.

A New 3D Super-Resolution Microscopy with Nanoscale Precision

Recently, a research team led by Prof. Philip Tinnefeld combined various methods to achieve ultra-high resolution and a faster imaging process to study dense molecular structures. Here, pulsed-interleaved MINFLUX nanoscopy (pMINFLUX) and GET were combined with DNA-PAINT to obtain nanoscale precision in 3D high-resolution imaging. Notably, this new approach offers an axial resolution of under 0.3 nm.

Tinnefeld’s team previously developed the pMINFLUX method that uses graphene as an energy acceptor. This method measures the fluorescence intensity of molecules that were excited by laser pulses. pMINFLUX can effectively distinguish between the lateral distances with 1 nm resolution. To measure the axial distance, the fluorescence intensity of the molecule was measured on the basis of its distance from graphene.

In GET-pMINFLUX nanoscopy, each photon is synergistically used for xy and z localization to gain optimal information about the object. To be precise, MINFLUX is associated with outstanding localization precision in xy, while GET offers ultra-high z-localization at close proximity to the coverslip surface. The new technique utilizes DNA origami nanopositioners, fluorescent molecules, and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) for 3D imaging at nanoscale dimensions. DNA-PAINT offers the switching mechanism that enables a transition from super-localization to super-resolution.

To elevate the binding kinetics, local PAINT (L-PAINT) was combined with GET-pMINFLUX. Scientists explained that longer concentrator sequences fixed imager strands to the specific region of interest while scanning. L-PAINT offered imaging of docking sites in less than 2 seconds with high resolution in 3D. It also enabled rapid tracking of binding trajectory in high resolution. L-PAINT also overcomes the shortcomings associated with fast imaging, such as thermal drift, and can also be applied to study dense molecular clusters.

Importantly, L-PAINT can be applied beyond DNA nanostructures. For instance, this technique could be applied for cell imaging with similar docking sites, which have a contradictory association with imager sequences and the concentrator. 

Scientists pointed out that the limitations related to the photon budget of the dye could be overcome by the slow exchange of the imager strands with weakened concentrator sequences. It must be noted that shortcomings related to the photon budget of the dye were not associated with MINFLUX, but with a less photon-efficient camera-based localization strategy.

Taken together, GET-pMINFLUX nanoscopy is an extremely powerful tool that can provide axial information by using a glass coverslip containing graphene. In the future, this nanoscopy could be used to study cellular membranes, macromolecular complexes, and artificial bilayers, with nanoscale 3D precision. Tinnefeld stated, “Our combination of GET-pMINFLUX and L-PAINT enables us to investigate structures and dynamics at the molecular level that are fundamental to our understanding of biomolecular reactions in cells.”


Zähringer, J. et al. (2023) Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy. Light Science and Applications, 12, 70.


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Dr. Priyom Bose

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.


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