Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.
Bacteriophage lambda-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 lambda-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 lambda-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 approximately 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 lambda-DNA shown here.
van 't Hoff
Marcel
M
INSERM, U603, Paris, France.
Reuter
Marcel
M
Dryden
David T F
DT
Oheim
Martin
M
D010582:Bacteriophage lambda / Q000737:chemistry*; D001583:Benzoxazoles / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D007364:Intercalating Agents / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D011806:Quinolinium Compounds / Q000032:analysis
D001583:Benzoxazoles; D004279:DNA, Viral; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium