Focus image scanning microscopy for sharp and gentle super-resolved microscopy

Giorgio Tortarolo, Alessandro Zunino, Francesco Fersini, Marco Castello, Simonluca Piazza, Colin J. R. Sheppard, Paolo Bianchini, Alberto Diaspro, Sami Koho and Giuseppe Vicidomini ()
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Giorgio Tortarolo: Istituto Italiano di Tecnologia
Alessandro Zunino: Istituto Italiano di Tecnologia
Francesco Fersini: Istituto Italiano di Tecnologia
Marco Castello: Istituto Italiano di Tecnologia
Simonluca Piazza: Istituto Italiano di Tecnologia
Colin J. R. Sheppard: Nanoscopy & NIC@IIT, Istituto Italiano di Tecnologia
Paolo Bianchini: Nanoscopy & NIC@IIT, Istituto Italiano di Tecnologia
Alberto Diaspro: Nanoscopy & NIC@IIT, Istituto Italiano di Tecnologia
Sami Koho: Istituto Italiano di Tecnologia
Giuseppe Vicidomini: Istituto Italiano di Tecnologia

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: Abstract To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

Date: 2022
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DOI: 10.1038/s41467-022-35333-y

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