title: Automation of a single-DNA molecule stretching device.
abstract: We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however, they should also be general to other applications of fluorescence-based microfluidics.
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Kristian Tølbøl
authors.initials: KT
authors.lastname: Sørensen
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, København, Denmark.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Lopacinska
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, København, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Tommerup
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, København, Denmark.
authors.forename: Asli
authors.initials: A
authors.lastname: Silahtaroglu
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Kristensen
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Marie
mesh_terms: D057205:Automation, Laboratory / Q000295:instrumentation* / Q000379:methods; D004247:DNA; D004867:Equipment Design; D005453:Fluorescence; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods; D011312:Pressure
chemical_list: D004247:DNA