FluoroTensor: Identification and tracking of colocalised molecules and their stoichiometries in multi-colour single molecule imaging via deep learning

Max F K Wills; Carlos Bueno Alejo; Nikolas Hundt; Andrew J Hudson; Ian C Eperon

doi:10.1016/j.csbj.2024.02.004

FluoroTensor: Identification and tracking of colocalised molecules and their stoichiometries in multi-colour single molecule imaging via deep learning

Comput Struct Biotechnol J. 2024 Feb 8:23:918-928. doi: 10.1016/j.csbj.2024.02.004. eCollection 2024 Dec.

Authors

Max F K Wills^{1

2}, Carlos Bueno Alejo^{1

3}, Nikolas Hundt⁴, Andrew J Hudson^{1

3}, Ian C Eperon^{1

2}

Affiliations

¹ Institute for Structural and Chemical Biology, University of Leicester, UK.
² Department of Molecular and Cell Biology, University of Leicester, UK.
³ Department of Chemistry, University of Leicester, UK.
⁴ Department of Cellular Physiology, Ludwig-Maximilians-Universität München, Germany.

Abstract

The identification of photobleaching steps in single molecule fluorescence imaging is a well-established procedure for analysing the stoichiometries of molecular complexes. Nonetheless, the method is challenging with protein fluorophores because of the high levels of noise, rapid bleaching and highly variable signal intensities, all of which complicate methods based on statistical analyses of intensities to identify bleaching steps. It has recently been shown that deep learning by convolutional neural networks can yield an accurate analysis with a relatively short computational time. We describe here an improved use of such an approach that detects bleaching events even in the first time point of observation, and we have included this within an integrated software package incorporating fluorescence spot detection, colocalisation, tracking, FRET and photobleaching step analyses of single molecules or complexes. This package, known as FluoroTensor, is written in Python with a self-explanatory user interface.

Keywords: Colocalisation; Convolutional neural networks; Diffusion parameters; Single molecule imaging; Single particle tracking; Stoichiometries of protein fluorophores.