[PDF]    https://doi.org/10.3952/physics.2024.64.4.7

Open access article / Atviros prieigos straipsnis
Lith. J. Phys. 64, 259–275 (2024)

SINGLE PIXEL RECONSTRUCTION IMAGING: TAKING CONFOCAL IMAGING TO THE EXTREME
Simona Streckaitea,b, Dmitrij Frolova, Jevgenij Chmeliovb,c, Andrius Gelzinisb,c, Cristian Ilioaiaa, Sylvie Rimskyd, Rienk van Grondellee, Leonas Valkunasb, Andrew Galla, and Bruno Roberta
a Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
b Department of Molecular Compound Physics, Center for Physical Sciences and Technology, Saulėtekio 3, 10257 Vilnius, Lithuania
c Institute of Chemical Physics, Faculty of Physics, Vilnius University, Saulėtekio 9, 10222 Vilnius, Lithuania
d CIRB, Collège de France, Université PSL, CNRS, INSERM, 75005 Paris, France
e Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands
Email: bruno.robert@I2bc.paris-saclay.fr

Received 3 July 2024; accepted 29 July 2024

Light nanoscopy is attracting widespread interest for the visualization of fluorescent structures at the nanometre scale. Recently, a variety of methods have overcome the diffraction limit, yet in practice they are often constrained by the requirement of special fluorophores, nontrivial data processing, or a high price and complex implementation. Therefore, confocal microscopy, yielding a relatively low resolution, is still the dominant method in biosciences. It was shown that image scanning microscopy (ISM) with an array detector could improve the resolution of confocal microscopy. Here, we review the principles of the confocal microscopy and present a simple method based on ISM with a different image reconstruction approach, which can be easily implemented in any camera-based laser-scanning set-up to experimentally obtain the theoretical resolution limit of confocal microscopy. Our method, single pixel reconstruction imaging (SPiRI), enables high-resolution 3D imaging utilizing image formation only from a single pixel of each of the recorded frames. We achieve the experimental axial resolution of 330 nm, which was not shown before by basic confocal or ISM-based systems. The SPiRI method exhibits a low lateral-to-axial FWHM aspect ratio, which means a considerable improvement in 3D fluorescence imaging. As a demonstration of SPiRI, we present the 3D-structure of a bacterial chromosome with an excellent precision.
Keywords: fluorescence imaging, microscopy, point-spread function, super-resolution, confocal microscopy, bacterial chromosome

VAIZDŲ REKONSTRUKCIJA IŠ VIENO PIKSELIO: KONFOKALINIO VAIZDINIMO RIBA
Simona Streckaitėa,b, Dmitrij Frolova, Jevgenij Chmeliovb,c, Andrius Gelžinisb,c, Cristian Ilioaiaa, Sylvie Rimskyd, Rienk van Grondellee, Leonas Valkūnasb, Andrew Galla, Bruno Roberta

a Paryžiaus Saklė universitetas, Esonas, Prancūzija
b Fizinių ir technologijos mokslų centro Molekulinių darinių fizikos skyrius, Vilnius, Lietuva
c Vilniaus universiteto Fizikos fakulteto Cheminės fizikos institutas, Vilnius, Lietuva
d Paryžiaus mokslų ir literatūros tyrimų universitetas, Paryžius, Prancūzija
e Amsterdamo laisvojo universiteto Fizikos ir astronomijos fakultetas, Amsterdamas, Olandija

Šviesos nanoskopija sulaukia didelio susidomėjimo vizualizuojant fluorescencines struktūras nanometrų skalėje. Pastaruoju metu difrakcijos ribą įveikė įvairūs nanoskopijos metodai, tačiau praktiškai juos dažnai riboja specialių fluoroforų poreikis, netrivialus gautų duomenų apdorojimas arba didelė kaina bei sudėtingas techninis įgyvendinimas. Dėl šių priežasčių konfokalinė mikroskopija, pasižyminti palyginti maža skiriamąja geba, vis dar yra dominuojantis metodas biomoksluose. Anksčiau buvo parodyta, kad vaizdo skenavimo mikroskopija (Image Scanning Microscopy; ISM) su matriciniu detektoriumi gali pagerinti konfokalinės mikroskopijos skiriamąją gebą. Šiame darbe apžvelgiame konfokalinės mikroskopijos principus ir pateikiame paprastą metodą, pagrįstą ISM, taikant kitokį vaizdo suformavimo būdą. Šį metodą galima lengvai įgyvendinti bet kurioje vaizdinimo sistemoje, pagrįstoje lazeriniu skenavimu su kameros detekcija, ir eksperimentiškai pasiekti teorinę konfokalinės mikroskopijos skiriamosios gebos ribą. Mūsų metodas, vieno pikselio rekonstrukcijos vaizdinimas (Single Pixel Reconstruction Imaging; SPiRI), leidžia atlikti didelės raiškos 3D vaizdinimą, naudojant vaizdų formavimą tik iš vieno kiekvieno įrašyto kadro pikselio. Mes pasiekiame eksperimentinę 330 nm z plokštumos skiriamąją gebą, kuri anksčiau nebuvo parodyta įprastose konfokalinėse ar ISM pagrįstose vaizdinimo sistemose. SPiRI metodas pasižymi mažu xy plokštumos ir z plokštumos skiriamųjų gebų FWHM santykiu, o tai reiškia didelį 3D fluorescencinio vaizdo pagerėjimą. Siekdami pademonstruoti SPiRI, pristatome didelio tikslumo 3D bakterijų chromosomos struktūrą.


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