However, advances have primarily been made in lateral resolutions – in other words, in the two directions perpendicular to a microscope’s line of sight. The ability to image structures in the axial direction – directly along the microscope’s line of sight – has lagged behind. This has prevented researchers from gaining 3D images of nano-sized structures, which is limiting when studying the molecular structures of cells.

A team led by Jörg Enderlein at Georg August University has combined the latest advances in lateral imaging with a cutting-edge technique for axial imaging – based on mapping the positions of single fluorescent molecules. Their approach could enable researchers to create clear images in all three dimensions, down to the scale of single biomolecules.

The team’s work began with an innovative twist on an imaging technique based on Förster resonance energy transfer, or FRET. Through FRET, energy is transferred from one fluorescent molecule to another. Crucially, the efficiency of this transfer depends on the distance between molecules, which can be calculated by measuring the fluorescence lifetime of the energy-donating molecule. By ‘labelling’ molecular structures with these fluorescent molecules, FRET enables researchers to map out distances between structures with powerful accuracy. However, this could only be done to a limited resolution.

In 2014, Enderlein’s team released a study, where they substituted the FRET acceptor molecule with a metal surface. With this approach, named metal-induced energy transfer, or MIET, the researchers extended the range over which measurements could be made to more than 100 nanometres: some 10 times the reach of FRET. This allowed them to build up vividly detailed images of cell structures and membranes. 

Building on the success of MIET, and with the help of funding from the European Research Council, the team pushed the boundaries of the technology further by swapping the metallic surface with graphene: a single layer of carbon atoms with unique optical properties. Graphene made the energy transfer efficiency of a fluorescent donor molecule even more sensitive to its distance from the surface. Named graphene-induced energy transfer, or GIET, this new approach boosted MIET’s already impressive sensitivity further still, allowing the team to measure the thickness of cell membranes with sub-nanometre precision.

In 2021, the team released a new study combining the principles of MIET with the latest advances in lateral imaging. Named single-molecule localization microscopy, or SMLM, this technique is based on the idea that the centre of an individual fluorescent molecule can be localized with greater accuracy than the much larger width of its image in a microscope – allowing the lateral positions of fluorescent labels to be mapped out with pinpoint precision. Enderlein’s team suggested that SMLM would provide the perfect complement to MIET’s ability to resolve axial positions. By integrating MIET with two forms of SMLM named dSTORM and PAINT, the researchers could finally capture the intricate 3D architectures of living cells, without sacrificing clarity in any direction.

Through continued funding from the European Research Council, Enderlein’s team is hopeful that the resolution of their technique could improve further still. This could be done by integrating dSTORM with the team’s recently-developed GIET technology – boosting the technique’s sensitivity to axial distances of Angströms – just a fraction of a nanometre. This innovative technology offers researchers an unprecedented view of the biomolecular world, paving the way for a host of exciting new discoveries.