A critical requirement for understanding and diagnosing severe pathologies, such as cancers, neurodegenerative or cardiovascular diseases, consists in achieving an exact understanding of fundamental structures and processes in cells and tissues at optical resolutions beyond the diffraction barrier. Fluorescence based techniques for Super-Resolution Microscopy (SRM) have generated huge impact in many fields of science but exhibit nonetheless serious limitations. For example, they are not chemically sensitive and require fluorescent probes, which may generate artefacts.

Furthermore, in living cells, fluorescent labelling can influence the metabolic behaviour and locomotion of the cells, and lead to cytotoxicity and phototoxicity. Higher-Harmonic Generation Microscopies (HHG) have gained massive attention due to their superb capabilities to image unlabelled samples based on very valuable and efficient contrast mechanisms. Second Harmonic Generation Microscopy (SHG) and Third Harmonic Generation Microscopy (THG) combined with (auto)fluorescence can enable a powerful 3D visualization of the extracellular matrix, of cell morphology or of subcellular components and organisation, which are fundamental for a thorough understanding of cell–tissue interaction and function. However, reliable SHG and THG beyond the diffraction barrier is still a challenge for the optics, photonics and high-resolution imaging communities, and no commercial system for super-resolution HHG currently exists on the market.

The main objective of HARMOPLUS is to demonstrate SHG and THG at sub-diffraction resolution using a novel approach inspired from the Re-scan Confocal Microscope (RCM). HARMOPLUS will build and test a prototype system for Re-scan HHG (incorporating also regular RCM) which will provide 40% improved resolution, and 4x increased sensitivity compared to the current state-of-the-art commercial systems for HHG imaging. From the proof-of-concept prototype system and results to be achieved in the frame of HARMOPLUS at TRL2-3, Confocal.nl expects to launch the product on the international markets in 3 years.

New publication: Super-resolution re-scan second harmonic generation microscopy. The Proceedings of the National Academy of Sciences (PNAS)
Link to the publication.