Issue 35, 2009
From the journal:

Physical Chemistry Chemical Physics

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques†

Marcel van ’t Hoff,‡abc   Marcel Reuter,‡d   David T. F. Drydend  and  Martin Oheim*abc  

* Corresponding authors

a INSERM, U603, Paris, France

b CNRS UMR 8154, Paris, France

c University Paris Descartes, Laboratory of Neurophysiology & New Microscopies, Paris, France
E-mail: martin.oheim@parisdescartes.fr
Fax: +33 1 42 86 41 51
Tel: +33 1 42 86 42 21

d School of Chemistry, West Mains Road, University of Edinburgh, Edinburgh, Scotland, UK

Abstract

Bacteriophage λ-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single λ-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and λ-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching ∼1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of λ-DNA shown here.

Graphical abstract: Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques

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Article information

DOI
https://doi.org/10.1039/B823155A
Article type
Paper
Submitted
05 Jan 2009
Accepted
01 May 2009
First published
29 Jun 2009

Phys. Chem. Chem. Phys., 2009,11, 7713-7720

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Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques

M. van ’t Hoff, M. Reuter, D. T. F. Dryden and M. Oheim, Phys. Chem. Chem. Phys., 2009, 11, 7713 DOI: 10.1039/B823155A

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