29 Jul 2025
Image reconstruction outperforms conventional methods for study of cell activity, drug effects.
A project at the Italian Institute of Technology (IIT) has developed a method able to carry out simultaneous super-resolution imaging and optical sectioning in laser scanning microscopy.
Described in Nature Photonics, the technique allows scientists to see and photograph biological samples in all their complexity, said the team, obtaining clear and detailed images.
The study, from IIT’s Molecular Microscopy and Spectroscopy group and funded within the European BrightEyes research project, was designed to address a specific issue: obtaining extremely sharp and detailed images of thick and complex biological samples.
BrightEyes, running from 2019 until February 2025, developed a novel single-photon detector array to study molecular interactions in living multicellular environments, as part of a protocol for continuous real-time tracking of individual biomolecules and decoding of their dynamics and interactions.
“What we did was rethink the way microscopes measure the light that hits the samples under observation, improving both the spatial resolution and the contrast when studying thick tissues, where background light would normally overpower their structure and create noise in the images,” said IIT’s Giuseppe Vicidomini.
The team developed a method for taking single-plane image data and reconstructing super-resolution optical sectioning, naming the technique super-resolution sectioning image scanning microscopy (ISM), or s2ISM.
In ISM a confocal laser scanning microscopy approach is combined with a detector array, which inherently encodes axial information within its imaging; but the complexity involved in recovering that axial data has been considerable. The IIT approach involved a conceptual change in how the ISM operation was framed by the researchers, who treated it as an exercise in structured illumination.
Cell interactions in real time
IIT designed an instrument in which a small array of sensors captures both the light at the point where it hits a tissue sample, and the way the light then spreads within that sample. Once this information is recorded, a reconstruction algorithm identifies the path of the light through the sample, as a way to produce sharper and better-sectioned images without losing signal quality.
“The optical microscope used is equipped with an array of SPAD (single-photon avalanche diode) detectors, capable of detecting the arrival of individual photons with very high spatial and temporal precision,” said Alessandro Zunino from IIT.
“This characteristic not only improves the resolution and optical sectioning, but also enables advanced techniques such as fluorescence lifetime imaging, which are fundamental to explore molecular dynamics in living tissues and to provide functional as well as structural information.”
Trials applying the s2ISM method to cell cultures and zebrafish embryos confirmed that it could outperform conventional image reconstruction techniques, and could be extended to any laser scanning microscopy technique.
“Potential applications are numerous: from studying brain tissue, tumors, organoids, and other complex biological systems, to direct observation of cellular life to understand disease progression,” commented IIT. “In the pharmaceutical field, the technique could be used to visualize in real time how drugs interact with living biological tissues, speeding up and enhancing the accuracy of the discovery of new treatments and therapies.”