Cappellari, M Victoria;
Marcano-García, Luis F;
Simoncelli, Sabrina;
Aramendía, Pedro F;
(2022)
Determination of association equilibrium constant from single molecule fluorescence localization microscopy.
Photochemical and Photobiological Sciences
, 21
pp. 1751-1760.
10.1007/s43630-022-00254-8.
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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.
| Type: | Article |
|---|---|
| Title: | Determination of association equilibrium constant from single molecule fluorescence localization microscopy |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1007/s43630-022-00254-8 |
| Publisher version: | https://doi.org/10.1007/s43630-022-00254-8 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Equilibrium constant, FRET, Fluorescence, Hybridization, Super resolution |
| UCL classification: | UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology UCL > Provost and Vice Provost Offices > UCL BEAMS UCL |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10152948 |
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