Anna Gaugutz und Gerhard Schütz im Labor
Photo Credit: Technische Universität Wien
Scientific Frontline: "At a Glance" Summary
- Main Discovery: Researchers developed a novel hybrid microscopy technique that maps the local refractive index of biological samples with resolution capabilities significantly below the diffraction limit of light.
- Methodology: The team combined single-molecule localization microscopy with atomic force microscopy; by independently measuring the sample's physical topography, they inverted standard optical errors to calculate the precise refractive index based on the variable size of light spots emitted by fluorescent markers.
- Key Data: The technique resolves structural details far smaller than the wavelength of visible light, enabling the precise quantification of local variations such as water content within collagen fibers.
- Significance: This innovation transforms a persistent source of optical error—variable refractive index—into a high-precision measurement parameter, successfully bridging physical measurement techniques with microbiological structural analysis.
- Future Application: Immediate applications focus on analyzing hydration levels in collagen-rich tissues and non-invasively assessing the chemical state of biological samples for disease research.
- Branch of Science: Biophysics and Applied Physics
- Additional Detail: The breakthrough emerged serendipitously when researchers reversed their original goal of correcting image distortions caused by the variable optical properties of samples, realizing the distortion itself contained valuable data.
A remarkable success has been achieved at TU Wien. By combining two fundamentally different microscopy techniques, researchers can now measure the optical properties of a sample with pinpoint accuracy.
The original goal was to investigate biological samples on a molecular scale – but this soon led to stubborn technical problems. Eventually, however, the researchers realized that the very source of their frustrating measurement inaccuracies – the variable refractive index of the sample – could itself be determined precisely. When two fundamentally different microscopy methods are combined, this former source of error turns into a highly informative measurement result.
In this way, and almost by chance, researchers at TU Wien developed a novel microscopy technique that allows the refractive index of biological samples to be measured at a resolution far below what conventional light microscopy theory would seem to allow.
The trick behind resolution beyond the wavelength of light
What happens if you try to image two molecules whose separation is smaller than the wavelength of light? You will not see two distinct points, but a single blurred spot of light – the images of the two molecules overlap, no matter how precise the microscope is.
There is, however, a way out: a so-called single-molecule microscopy. Special fluorescent molecules are deliberately introduced into the sample, where they emit light at different points in time. Each molecule produces a small disk of light on the camera. By determining the center of this disk, the position of the molecule can be pinpointed with great accuracy. Even if another molecule lies within the same disk: as long as the molecules light up one after the other and can be measured separately, both can be imaged precisely. While their images would simply blur together in a conventional microscope image, this method enables extremely high-resolution imaging.
“The light disks we measure in this way are not always the same size,” says Prof. Gerhard Schütz from the Institute of Applied Physics at TU Wien. “The size of the light disk depends, for example, on how far the molecule is from the focal plane of the camera.” Initially, the researchers wanted to use this effect as a source of useful information: if the distance of a molecule could be inferred from the size of the light disk, it should in principle be possible to reconstruct a three-dimensional image. But it soon became clear that the situation was more complicated.
Is it the distance – or the refractive index?
“The problem is that the size of the light disk also depends on the refractive index of the material,” explains Gerhard Schütz. Not all materials allow light to propagate at the same speed – and this is precisely the effect that causes light to be deflected by prisms or lenses. Thus, there are two parameters that influence the measured light spot: distance and refractive index.
But what if this difficulty could be turned into an advantage? “We decided to reverse the problem,” says Gerhard Schütz. “We measure the three-dimensional structure of our sample in a completely different way, using an atomic force microscope. We can then use the optical data to calculate the refractive index at each specific location in the sample.”
A new measurement method for biological materials research
In collaboration with the Medical University of Innsbruck, the team at TU Wien developed a technique that makes it possible to measure the refractive index of biological samples on a length scale far below the wavelength of light.
“This is particularly exciting when it comes to collagen in biological tissue,” says Gerhard Schütz. “Collagen can absorb different amounts of water, and this directly affects its refractive index. With our method, we can now determine, with high spatial precision, how much water is present at different locations. We can also obtain information about the chemical state of the tissue that was previously not directly accessible.”
The result – triggered by an almost accidental discovery – is a new link between physical measurement techniques and microbiological research.
Funding: The research was funded by the Austrian Science Fund (FWF) and the Vienna Science and Technology Fund (WWTF), and emerged from a collaboration between the Institute of Applied Physics and the Institute of Lightweight Design and Structural Biomechanics at TU Wien, as well as the Institute of Biomedical Physics at the Medical University of Innsbruck.
Published in journal: ACS Nano
Title: Refractive Index Mapping below the Diffraction Limit via Single Molecule Localization Microscopy
Authors: Simon Jaritz, Lukas Velas, Anna Gaugutz, Manuel Rufin, Philipp J. Thurner, Orestis G. Andriotis, Julian G. Maloberti, Simon Moser, Alexander Jesacher, and Gerhard J. Schütz
Source/Credit: Technische Universität Wien
Reference Number: phy012926_01
.jpg)