Optics+Photonics Showcase
daily coverage of the optics & photonics industry and the markets that it serves
Menu
Research & Development

LMU Munich develops microscopy method that beats resolution limit

15 Feb 2024

“MINFLUX” super-resolution microscopy can track molecules with localization at 1nm precision.

Processes in the human body are shaped by the interaction of various biomolecules, such as proteins and DNA. These processes take place in a range of often just a few nanometers. They can therefore no longer be observed using fluorescence microscopy, whose resolution limit is around 200 nm due to light diffraction.

If two dyes used to mark biomolecules are closer together than this optical boundary, their fluorescence can no longer be distinguished under the microscope. However, since this is used to localize the dyes, correct position determination is impossible.

Classically, the resolution limit in super-resolution microscopy methods is circumvented by making the dyes blink and literally turning their fluorescence on and off again. In this way, the fluorescence is separated in time and can therefore be distinguished, which enables localizations below the classic resolution limit.

A solution: MINFLUX multiplexing

However, for applications in which fast dynamic processes are examined, this trick has a crucial disadvantage: the blinking ensures that several dyes cannot be localized at the same time. This significantly degrades the temporal resolution when studying dynamic processes that involve multiple biomolecules.

Under the leadership of LMU (Ludwig-Maximilians University, Munich, Germany) chemist Professor Philip Tinnefeld and in cooperation with Professor Fernando Stefani (Buenos Aires), LMU researchers have now developed an elegant approach to solve this problem using pMINFLUX multiplexing. The method was recently published in the journal Nature Photonics.

MINFLUX is a super-resolution microscopy method that enables localizations with precisions of just one nanometer. In contrast to conventional MINFLUX, pMINFLUX records the time difference between the excitation of the dyes with a laser pulse and the resulting fluorescence in sub-nanosecond resolution.

In addition to their localization, this allows insights into a fundamental property of fluorescent dyes: their fluorescence lifetime. This describes how long it takes on average for a dye molecule to fluoresce after it has been excited.

“The fluorescence lifetime depends on the dye used,” said Fiona Cole, first author of the publication. “We used differences in fluorescence lifetime when using different dyes to assign fluorescence to different dye molecules without the need for blinking and the associated temporal separation.”

The researchers adapted the localization algorithm and incorporated a multi-exponential fit model to achieve the desired separation. “This allowed us to determine the position of several dyes at the same time and thus investigate fast dynamic processes between several molecules with nanometer-level precision,” added Jonas Zähringer, also first author.

The researchers demonstrated their method by accurately tracking two DNA strands as they move between different positions on a DNA origami nanostructure, separating translational and rotational movements of a DNA origami nanostructure, and measuring the distance between antigen binding sites of antibodies.

“But this is just the beginning,” said Philip Tinnefeld. “I am sure that pMINFLUX multiplexing with its high temporal and spatial resolution will provide new insights into protein interactions and other biological phenomena in the future.”

LaCroix Precision OpticsHÜBNER PhotonicsCeNing Optics Co LtdCHROMA TECHNOLOGY CORP.Iridian Spectral TechnologiesUniverse Kogaku America Inc.SPECTROGON AB
© 2024 SPIE Europe
Top of Page