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Determination of association equilibrium constant from single molecule fluorescence localization microscopy

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Abstract

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

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Acknowledgements

MVC and LFMG are fellows and PFA is staff member of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, and full professor (Universidad de Buenos Aires). We thank Dr. Leonardo Lizarraga (CIBION, CONICET) for the AFM measurements. The work was financed by research grants from CONICET (PIP0626) and ANPCyT (PICT 2014-3634). SS acknowledges financial support from the Royal Society through a Dorothy Hodgkin fellowship (DHF\R1\191019).

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Authors and Affiliations

  1. Centro de Investigaciones en Bionanociencias -‘‘Elizabeth Jares-Erijman’’ (CIBION), CONICET, Godoy Cruz 2390, 1425, Ciudad de Buenos Aires, Argentina

    M. Victoria Cappellari, Luis F. Marcano-García & Pedro F. Aramendía

  2. London Centre for Nanotechnology and Department of Chemistry, University College London, London, WC1H 0AH, UK

    Sabrina Simoncelli

  3. Departamento de Química Inorgánica, Analítica y Química Física. Facultad de Ciencias Exactas Y Naturales, Universidad de Buenos Aires, 1428, Ciudad de Buenos Aires, Argentina

    Pedro F. Aramendía

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  1. M. Victoria Cappellari
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Correspondence to Pedro F. Aramendía.

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This publication is dedicated to Prof. Silvia E. Braslavsky, a pioneer in photobiology and photobiophysics, on the occasion of her 80th birthday.

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Cappellari, M.V., Marcano-García, L.F., Simoncelli, S. et al. Determination of association equilibrium constant from single molecule fluorescence localization microscopy. Photochem Photobiol Sci 21, 1751–1760 (2022). https://doi.org/10.1007/s43630-022-00254-8

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  • DOI: https://doi.org/10.1007/s43630-022-00254-8

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