Jump to main content
Jump to site search

Issue 35, 2009
Previous Article Next Article

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

Author affiliations

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

Back to tab navigation

Supplementary files

Publication details

The article was received on 05 Jan 2009, accepted on 01 May 2009 and first published on 29 Jun 2009


Article type: Paper
DOI: 10.1039/B823155A
Citation: Phys. Chem. Chem. Phys., 2009,11, 7713-7720

  •   Request permissions

    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

Search articles by author

Spotlight

Advertisements