Alberto Diaspro is Full Professor of Applied Physics at the Physics Department of the University of Genoa, PI at the Italian Institute of Technology, affiliate at IBF-CNR, full academic of the Ligurian Academy of Sciences and Letters, member of the Scout di Letture e Conversazioni Scientifiche, Genova. Active in European and national projects, he has over 400 scientific articles with 25000 citations (H = 67). AD designed and realised the 1st Italian multiphoton microscope (1999) and a hybrid artificial "nanobiorobot" (2000). He directed the design and realisation of the 1st Italian nanoscopy architecture at IIT (2008). He co-founded the Genoa Instruments start-up in 2019, and recently launched "The artificial microscope", EOS Dijon (2023). He is developing an intelligent quantum optical microscope. He is scientific responsible of projects within the PNRR, Next Generation EU funds, “SeeLife” (B53C22001810006), PNRR CN3, and National Quantum Technologies-NQSTI. He received the Emily M. Gray Award from the Biophysical Society for "significant contributions to education in biophysics" and the Award for Scientific Communication from the Italian Physical Society in 2019. AD was President of EBSA, OWLS, and of the Scientific Council of Festival of Science, Genoa. He is SPIE and EOS Fellow, IEEE and OSA senior member. He coordinates the ALM node of Eurobioimaging. AD founded the Nanoscale Biophysics subgroup of the Biophysical Society. AD is editor-in-chief of Microscopy Research and Technique, President of the Italian Society of Pure and Applied Biophysics, SIBPA; Co-Director of the International School of Biophysics «Antonio Borsellino» "Ettore Majorana Foundation and Centre for Scientific Culture, Erice. AD received the Gregorio Weber Award for excellence in fluorescence studies in 2022. On 2024, AD has been appointed Ian Venice as Academic of the “Istituto Veneto di Scienze, Lettere ed Arti (IVSLA)” , and as Cavaliere dell’Ordine al “Merito della Repubblica Italiana".
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Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.
ISM can be performed in fluorescence, bright field or interference microscopy. Several different implementations have been described, with associated advantages and disadvantages. In two-photon microscopy, the illumination and detection point spread functions are very different. This is also the case when using pupil filters or when there is a large Stokes shift.
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Two-photon excitation and confocal microscopy investigation of nanostructured polyelectrolyte shells
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