Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations.

Maged F Serag,Satoshi Habuchi,Maram Abadi

doi:10.1038/ncomms6123

Maged F Serag, Satoshi Habuchi + Show 1 more

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https://doi.org/10.1038/ncomms6123

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Abstract

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

Highlights

Background subtractionNoise removalForward and Backward superimposition Ai ΔAi Ai+1Cumulative area difference that occurs during the time lag Δt Data averagingD = 〈ΔAi 〉 4Δt ti superimposition
We further extend the potential usefulness of our approach by analysing the diffusion and conformational dynamics of double-strandedDNA of different lengths and topological forms, measurements that are critically sensitive to molecular size and conformational changes
We validate our approach experimentally by comparing the distribution of diffusion coefficients of approximately 100 cross-linked fluorescent polystyrene nanospheres with a mean diameter of 0.19 mm suspended in 1 mM Tris buffer measured by means of single-molecule fluorescence imaging and analysed by both the CA and single-molecule localization and tracking (SMLT)-mean square displacement (MSD) methods

Summary

Introduction

We validate our approach experimentally by comparing the distribution of diffusion coefficients of approximately 100 cross-linked fluorescent polystyrene nanospheres with a mean diameter of 0.19 mm suspended in 1 mM Tris buffer measured by means of single-molecule fluorescence imaging (see ‘Methods’ section) and analysed by both the CA and SMLT-MSD methods. The distribution of the diffusion coefficient obtained by the SMLT-MSD analysis can be reproduced reasonably by a theoretical statistical probability distribution of diffusion coefficients in a homogeneous environment (see ‘Methods’ section and Supplementary Fig. 5)

Methods

Results

Conclusion