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.
Originalsprog | Engelsk |
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Artikelnummer | 063702 |
Tidsskrift | Review of Scientific Instruments |
Vol/bind | 86 |
Udgave nummer | 6 |
Sider (fra-til) | 1-6 |
Antal sider | 6 |
ISSN | 0034-6748 |
DOI | |
Status | Udgivet - jun. 2015 |