image: a, pMINFLUX interrogates the position of a fluorophore with multiple spatially displaced doughnut beams and yields 2D fluorescence lifetime images with nanometer precision. b, Graphene provides a measure for the axial distance to graphene. The fluorescence lifetime shortens, the closer a fluorophore is to graphene. c, Combining the lateral information of pMINFLUX with the axial graphene distance information yields 3D localizations. GET-pMINFLUX yields photon efficient localizations with nanometer precision. This enables L-PAINT. The schematic of the DNA origami structure has a DNA-pointer protruding. The fluorophore modified DNA-pointer can transiently to one of three binding sites spaced with 6 nm. Within 2 s this dense structure is with nanometer precision localized in 3D by combining L-PAINT and GET-pMINFLUX. view more
Credit: by Jonas Zähringer, Fiona Cole, Johann Bohlen Florian Steiner, Izabela Kamińska, Philip Tinnefeld
Only a few years ago, a resolution limit in optical microscopy that seemed fundamental was broken, leading to the award of the Nobel Prize in Chemistry in 2014. Since then, there has been another quantum leap in the field of super-resolution microscopy, pushing the resolution limit to the molecular dimension (1 nm).
Scientists led by Professor Philip Tinnefeld (LMU) have now succeeded in combining 2D MINFLUX microscopy with a new method for axial resolution. This combination, which exploits special properties of the 2D material graphene, enables an axial precision of less than 3 angstroms.
The MINFLUX method interrogates the location of each molecule by placing a laser focus near the molecule. The measured fluorescence intensity serves as a measure of the distance of the molecule from the center of the laser focus. By placing the center of the laser focus successively at different positions relative to the molecule, the exact molecule position can be obtained by triangulation. With the pMINFLUX (pulsed MINFLUX) developed in the Tinnefeld and Stefani lab in 2020, the 2D microscopy has already been simplified. Here, the use of pulsed laser not only simplifies lateral super-resolution microscopy, but also intrinsically gives access to the fluorescence lifetime.
"With the combination of pMINFLUX and graphene, the information of a photon is used in the best possible way." explains Philip Tinnefeld. "Here, pMINFLUX is synergistically combined with graphene energy transfer (GET) to enable precisions so far unreached for fluorescence microscopy."
Due to GET, the fluorescence lifetime is distance-dependent, thus a change in fluorescence lifetime can be converted to axial distance from graphene. As a result, GET-pMINFLUX yields 3D localizations with axial precisions of less than 3 Angstroms.
"The high precisions achievable with GET-pMINFLUX enable the development of methodologically new approaches to improve super-resolution microscopy which were previously not realizable." explains Jonas Zähringer, first author of the publication. "Typically, the entire photon budget of a dye is needed to localize a position with 1 nm precision. However, GET-pMINFLUX is so photonefficient that the photon budget can be split among multiple positions and still yield nanometer precise localizations."
Using DNA nanotechnology, the researchers developed L-PAINT (local PAINT), a new approach to increase the speed of super-resolution microscopy. In contrast to DNA-PAINT, a technique that enables super-resolution by binding and unbinding a DNA strand labeled with fluorescent dye, L-PAINT has two binding sequences. For this purpose, the researchers designed a binding hierarchy so that the L-PAINT DNA strand binds longer on one side. This allows the other end of the strand to scan for binding sites with a high local concentration.
"The high local concentration not only increases the speed, but also enables scanning of dense clusters, faster than disturbances due to thermal drift." Philip Tinnefeld explains. "The combination with GET-pMINFLUX and L-PAINT allows us to study structures and dynamics at the molecular level, which are fundamental for our understanding of cellular biomolecular reactions."
Journal
Light Science & Applications