Researchers make fluorescence microscopy twice as precise

Breaking new ground 9. jun 2021 2 min Senior Researcher Kim Mortensen Written by Kristian Sjøgren

Research in Denmark has helped to boost fluorescence microscopy. Now researchers can see much more precisely how small proteins cross the intestinal wall.

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Fluorescence microscopy is one of the most important tools medical research uses to determine whether medicine taken orally is absorbed through the intestinal wall.

If not, the medicine will not work.

However, fluorescence microscopy of individual molecules has been quite imprecise. But now researchers in Denmark seem to have considerably improved the precision, thereby enabling much better insight into what individual molecules do when they pass through the gut and into the bloodstream.

The discovery may help to elucidate an area about which very little is known today.

“Determining how a pharmaceutical peptide crosses the intestinal wall requires knowledge about its exact location relative to the intestinal wall and possible interactions with other molecules in its path. Our new method for fluorescence microscopy is twice as precise as the methods used so far,” explains a researcher behind the discovery, Kim Mortensen, Senior Researcher, Department of Health Technology, Technical University of Denmark, Kongens Lyngby.

The research has been published in Communications Physics.

Reducing the margin of error in fluorescence microscopy

In fluorescence microscopy, researchers place a fluorescent molecule on the substances they want to examine in a given tissue, such as tiny fragments of protein in intestinal tissue.

When the researchers fire a laser beam at the samples and study them under a microscope, they can see where the molecules light up in the tissue and thus determine their location.

However, the challenge with this technique is that the fluorescent molecules are much smaller than the detected spot of light. The reason for this is diffraction, and this means that researchers cannot determine the exact location of the molecule in the illuminated spot.

Although researchers use numerous techniques to get more precise answers, they are constrained by the limited intensity of the signal emitted by the fluorescent molecules.

“We cannot be certain of the exact location of the molecule within the illuminated spot. We need to confirm this to specify where and how a peptide crosses the cell membrane. In other words, ideally, the image resolution must be comparable to the actual size of the molecules of interest,” says Kim Mortensen.

Exposing the sample to structured laser light

To address the problem, the researchers modified the excitation light path of the laser directed at a sample.

The researchers used interference of laser beams for generating the structured illumination that optimized the differences and transitions between light and dark regions.

This means that if a molecule is located somewhere in the illumination profile where there is light, it will light up, but it will not light up in a dark location. The amount of light observed depends on where the molecule is located in relation to the illumination profile. The researchers then phase-shift the profile and combine the two images of their sample into one. This brings them closer to the exact location of the molecule.

The result is an image with four times the information than could previously be obtained by using conventional fluorescence microscopy.

“Combining the images still shows the large spot of light, but we have the information from both the location of the spot and from the intensity of the spot. We can then analyse the information to determine twice as precisely where the molecule is actually located within the light spot,” explains Kim Mortensen.

More research ahead

Kim Mortensen says that the researchers have only shown that extracting more information from fluorescence microscopy images of individual molecules is technically and mathematically possible but that they will soon use the technique on actual tissue samples.

In addition, the researchers have only shown that this technique works in two dimensions, but the plan is to demonstrate that it also works in 3D.

“The common task of our research centre is that we want to study the mechanisms that are involved when medicine crosses the intestinal wall. We have come closer to being able to do that now,” says Kim Mortensen.

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