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Luciano Masullo

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Single-molecule localization with molecular-scale precision by raster scanning a minimum of light

Luciano A. Masullo1,2 *, Alan M. Szalai1, Lucía F. Lopez1, Mauricio Pilo-Pais3, Guillermo P. Acuna3, and Fernando D. Stefani1,2

1- Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2390, C1425FQD Ciudad Autónoma de Buenos Aires, Argentina.2- Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Güiraldes 2620, C1428EHA Ciudad Autónoma de Buenos Aires, Argentina.3- Department of Physics, University of Fribourg, Chemin du Musée 3, Fribourg CH-1700, Switzerland.* Present affiliation: Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Planegg, Germany

Localization of single fluorescent molecules is key for physicochemical and biophysical measurements such as single molecule tracking and super-resolution imaging. Over the last two decades, several methods have been developed in which the position of a single emitter is interrogated with a sequence of spatially modulated patterns of light [1-6]. Among them, the recent MINFLUX [5] technique outstands for achieving a 10-fold improvement compared with wide-field camera-based single-molecule localization, reaching 1–2 nm localization precision at moderate photon counts. Because they were developed independently and present important differences in their experimental setups and data analysis routines, various single-molecule localization methods based on sequential structured illumination, such as MINFLUX [6], MINSTED [7], or Orbital Tracking [1], have been considered as fundamentally different or slightly related. In this work, we present a common framework [7] that offers conceptual order and provides insight into the essential components of all methods for single-molecule localization based on sequential structured illumination. Interestingly, our calculations show that top single-molecule localization performances can be obtained in any raster-scanning microscope with minor modifications (e.g., a vortex phase plate in the excitation path). We will present the first experimental implementation of the latter method, here called RASTMIN, that allows 1-2 nm localization precision by raster scanning a minimum of light[8]. In contrast to other methods such as MINFLUX, RASTMIN can be easily implemented in a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in the localization of single molecules and super-resolution imaging of DNA-origami structures using a home-built confocal microscope with only minor modifications.

1- J. Enderlein, Applied Physic B (2000), 2- V. Levi et al, Biochem. Soc. Trans (2003), 3- K. Kis-Petikova, Microsc. Res. Tech. (2004), 4- A. Germann et al, Optics Express (2014), 5- F. Balzarotti et al, Science (2017), 6- M. Weber et al, Nature Photonics (2021), 7- L. Masullo et al, Biophysical Reports (2022), 8- L. Masullo et al, Light: Science and Applications (2022).

Luciano Masullo
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