Combining pMINFLUX with graphene energy transport for precise 3D localizations. one Top: Schematic drawing of a DNA origami structure with a single dye placed 16 nm high on top of 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. si Fluorescence is split for each of four pulsed vortex-shaped interference beams focused on the sample arranged in a triangular pattern with the fourth beam placed in the center of the triangular structure. The star indicates the xy position of the dye molecule. do xy histogram of locating time bins. Hey Distribution of fluorescence lifetimes obtained from time bins. m Distribution of distances in graphene z calculated from fluorescence lifetimes of d). eat 3D localizations of the full fluorescence intensity trace using the 2D information of pMINFLUX and z-distances from the fluorescence lifetimes. The individual localizations are shown in black and on the sides the corresponding projections with binning of 1 nm for xy and 0.2 nm for z. Credit: Light: Science & Applications (2023). DOI: 10.1038/s41377-023-01111-8
Super-resolution microscopy methods are essential for revealing cell structures and molecular dynamics. Since the researchers surpassed the resolution limit of about 250 nanometers (while winning the 2014 Nobel Prize in Chemistry for their efforts), which was long considered absolute, microscopy methods have advanced rapidly.
Now a team led by LMU chemist Professor Philip Tinnefeld has made further progress by combining various methods, achieving the highest resolution in three-dimensional space and paving the way for a fundamentally new approach to faster imaging of dense molecular structures. The new method allows axial resolution below 0.3 nanometers.
The researchers combined the so-called pMINFLUX method developed by Tinnefeld’s group with an approach that uses special properties of graphene as an energy receiver. pMINFLUX is based on measuring the fluorescence intensity of molecules excited by laser pulses. The method makes it possible to distinguish their lateral distances with a resolution of just 1 nanometer.
Graphene absorbs the energy of a fluorescent molecule no more than 40 nanometers from its surface. The fluorescence intensity of the molecule therefore depends on its distance from the graphene and can be used to measure the axial distance.
a, pMINFLUX probes the position of a fluorophore with multiple spatially displaced donut bundles and yields 2D fluorescence lifetime images with nanometer precision. b, graphene provides a measure of the axial distance from graphene. The shorter the fluorescence lifetime, the closer a fluorophore is to graphene. c, Combining the lateral information of pMINFLUX with the graphene axial distance information yields 3D localizations. GET-pMINFLUX yields efficient photon localizations with nanometer precision. This enables L-PAINT. The schematic of the DNA origami structure has a DNA marker sticking out. The fluorophore-modified DNA marker can transiently to one of three binding sites within 6 nm. Within 2 seconds this dense structure is located with nanometer precision in 3D by combining L-PAINT and GET-pMINFLUX. Creation: Jonas Zähringer, Fiona Cole, Johann Bohlen Florian Steiner, Izabela Kamińska, Philip Tinnefeld
DNA-PAINT increases speed
Consequently, combining pMINFLUX with this so-called graphene energy transfer (GET) provides information about molecular distances in all three dimensions – and does so at the highest resolution achievable to date, below 0.3 nanometers . “The high precision of GET-pMINFLUX opens the door to new approaches to improve super-resolution microscopy,” says Jonas Zähringer, lead author of the paper.
The researchers also used it to further increase the speed of super-resolution microscopy. To this end, they used DNA nanotechnology to develop the so-called L-PAINT approach. Unlike DNA-PAINT, a technique that allows ultra-analysis by binding and unbinding a DNA strand labeled with a fluorescent dye, the DNA strand in L-PAINT has two binding sequences.
In addition, the researchers designed a binding hierarchy so that the L-PAINT DNA strand binds more to one side. This allows the other end of the strand to locally scan the positions of the molecules at a rapid rate.
“In addition to increasing speed, this allows dense clusters to be scanned faster than distortions resulting from thermal drift,” says Tinnefeld. “The combination of GET-pMINFLUX and L-PAINT enables us to probe structures and dynamics at the molecular level that are fundamental to understanding biomolecular reactions in cells.”
The findings are published in the journal Light: Science & Applications.
More information:
Jonas Zähringer et al, Combining pMINFLUX, graphene energy transfer and DNA-PAINT for 3D super-resolution nanometer-precision microscopy, Light: Science & Applications (2023). DOI: 10.1038/s41377-023-01111-8
Provided by the Ludwig Maximilian University of Munich
Reference: A super-resolution microscopy method for rapid differentiation of molecular structures in 3D (2023, 10 March) Retrieved 11 March 2023 from https://phys.org/news/2023-03-super-resolution-microscopy-method-rapid – diff.html
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