Smart molecules for better microscopy images

Multiphoton microscopy is used in biomedical research to study cells and tissues. Today, so-called two-photon microscopy is used to study processes within cells, but the technique has limitations in terms of image resolution.

Four-photon microscopy provides images with higher resolution. However, such instruments are very expensive and, when studying biological material, the powerful laser light required can damage samples.

“In this project, we have developed molecules to visualise molecular-level details and monitoring processes using the more common two-photon microscopy technique. These molecules have the capacity to achieve higher resolution than with four-photon microscopy, although two-photon microscopy is used,” says the project coordinator Joakim Andréasson, Professor at the Department of Chemistry and Chemical Engineering at Chalmers University of Technology.

He continues:

“In the long term, results from studies of this kind may provide new insights into diseases, pharmaceuticals and the very smallest building blocks of life.”

Twice as good resolution

The researchers synthesized the molecules by linking fluorescent dyes with so-called molecular photoswitches, small molecules that change their properties when illuminated. The idea was to demonstrate that this combination could be used to achieve improved resolution.

When cell samples stained with these molecules are examined using two-photon microscopy, the molecules behave as if they were illuminated with the light used in four-photon microscopy, but achieving twice as good resolution compared to the latter teqchnique.

“In a recently published study in Nature Communications, we showed that the molecules exhibit four-photon behaviour, even when using two-photon microscopy. Further studies are required to optimise the technique, particularly for investigations in biologically relevant environments, such as live cells,” says Joakim Andréasson.

Broad expertise a major factor of project's success

The “4 for 2” project was awarded €2.3 million over three years by the EU Pathfinder Open initiative. The project is a collaboration between four universities: Chalmers University of Technology, the University of Gothenburg, KU Leuven in Belgium and the University of Huelva in Spain. Joakim Andréasson describes the projects broad, combined expertise − ranging from specialists in organic chemistry, spectroscopy and photo physics to chemical biology −as its key strength and a major factor in its success.

“The substantial project funding also gave us the opportunity to focus on ensuring high quality results without time pressure. We have been able to recruit dedicated staff and allow the research the time it requires. Now that the project is drawing to a close, we can see that we have been very successful in delivering on what we promised in our grant proposal, while also generating additional results for future studies. This would, of course, be facilitated by continued funding, which we are currently actively seeking,” says Joakim Andréasson.

Selected publications from the project

• Exploiting negative photochromism to harness a four-photon-like fluorescence response with two-photon excitation. Published in Nature Communications.
• Heteroaryl iminothioindoxyl (HA-ITI) photoswitches via regioselective aza-Wittig synthesis: unifying red-shifted absorption, large E/Z band separation, and tunable thermal recovery. Publisherad in Chemical Science.