Washington, D.C., April 29, 2022 — A label-free microscopy technique developed by researchers at the University of Graz enables non-invasive, sub-diffraction-limited imaging of nanostructures. This technology combines Fourier plane polarimetry and multipole retrieval with laser scanning microscopy to reconstruct sub-diffraction-limited nanoparticle assemblies. It builds on the capabilities of laser scanning microscopy, where a light beam scans the sample and measures the returned light. In addition to measuring the brightness of light interacting with the sample, the newly introduced technique also measures parameters encoded in the light field. Beyond brightness, the phase, polarization, and scattering angle of light provide information about the sample. To capture the information stored in these features, the researchers examined the spatial resolution of light intensity and polarization. “The way the phase and polarization of light, together with its intensity, vary spatially incorporates details of the sample it has interacted with — much like the shadow of an object tells us about the object’s own shape,” said Professor Peter Banzer, who led the research. “However, most of this information is ignored if only the overall light power is measured after interaction.” The researchers used their method to study basic samples containing gold nanoparticles of different sizes. After scanning the region of interest on the sample, they recorded polarization-resolved and angle-resolved images of the transmitted light and evaluated the measured data using a custom algorithm. This algorithm constructs a model of the nanoparticle assembly that accurately reflects the measured data. It also provides researchers with information about the number of particles, as well as their positions and sizes. The team validated their experimental results by comparing them with benchmark scanning electron microscopy images. “Although the particles and their distances are far smaller than the resolution limit of many microscopes, our method is able to resolve them,” Banzer said. “Moreover, and more importantly, the algorithm can provide additional parameters about the sample, such as the precise size and position of the particles.” Most microscopy techniques that can resolve images beyond the diffraction limit, such as super-resolution microscopy, require samples to be modified with fluorescent labels. Super-resolution techniques that do not rely on any form of sample modification have limited ability to improve resolution. “Our new approach to laser scanning microscopy can bridge the gap between traditional microscopes with limited resolution and super-resolution techniques that require modification of the specimen under study,” Banzer said. “Compared to super-resolution techniques based on similar scanning methods, our approach is completely non-invasive, meaning it does not require injecting any fluorescent molecules into the sample prior to imaging.” Now, the researchers are adapting the method to enable its use with more complex samples. They believe the method’s capabilities can be expanded by adjusting the structure of light interacting with the sample and integrating artificial intelligence-based methods into the image processing steps. The team is also working on developing a camera capable of resolving polarization and phase information in addition to intensity. The development of this next-generation detection device is part of a European project called SuperPixels. Measuring the distribution of nanoparticles is a common task in science, and this technology can be used in many scientific disciplines to reconstruct complex nanostructures and structural arrangements. “Our method can help expand the microscopy toolbox for studying nanostructures in various samples,” Banzer said. “Our research once again demonstrates the key role that light structure can play in the field of optics and light-based technologies.”