Highest Accuracy in Three Dimensions – ScienceDaily

LMU researchers have developed a super-resolution microscopy method for the rapid discrimination of 3D molecular structures.

Super-resolution microscopy methods are essential for revealing cell structures and molecular dynamics. Since researchers surpassed the resolution limit of about 250 nanometers (and were awarded the 2014 Nobel Prize in Chemistry for their efforts), long considered absolute, microscopy methods have evolved rapidly. Now a team led by LMU chemist Professor Philip Tinnefeld has made further progress by combining the different methods, achieving the highest resolution in 3D space and paving the way for a fundamentally new approach for faster imaging of dense molecular structures. The new method allows for axial precision of less than 0.3 nm.

The researchers combined the so-called pMINFLUX method developed by Tinnefeld’s team with an approach that uses special properties of graphene as an energy receiver. pMINFLUX is based on measuring the fluorescence intensity of particles excited by laser pulses. This method makes it possible to distinguish their lateral distances with an accuracy of only 1 nm. Graphene absorbs the energy of a fluorescent molecule no more than 40 nanometers away from its surface. Therefore the fluorescence intensity of a molecule depends on its distance from the graphene and can be used to measure the axial distance.

DNA-PAINT increases speed

Thus, the combination of pMINFLUX and what is called graphene energy transfer (GET) provides information about molecular distances in all three dimensions – and does so at the highest resolution achievable to date of less than 0.3 nanometers. “The high resolution 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 this to increase the speed of super-resolution microscopy. To this end, they have relied on DNA nanotechnology to develop what is called the L-PAINT approach. Unlike DNA-PAINT, which is a technology that enables superior fidelity by ligating 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 splicing hierarchy so that the L-PAINT DNA strand binds longer on one side. This allows the other end of the thread to scan the molecule positions locally at a rapid rate.

“In addition to the increased speed, this allows dense clumps to be cleared faster from distortions caused by thermal drift,” says Tinnefeld. “The combination of GET-pMINFLUX and L-PAINT enables us to investigate structures and dynamics at the molecular level that are central to our understanding of biomolecular interactions in cells.”

Source link

Related Posts