Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4868

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Fluorescence Imaging with One-Nanometer Accuracy (FIONA) of Cy3-DNA under Deoxygenation Conditions

Paul R. Selvin1, Tyler Lougheed, Melinda Tonks Hoffman, Hyokeun Park, Hamza Balci, Benjamin H. Blehm, and Erdal Toprak

Biophysics Center and Physics Department, University of Illinois, Urbana, IL 61801, USA

1Corresponding author (selvin{at}uiuc.edu)


INTRODUCTION

FIONA, short for fluorescence imaging with one-nanometer accuracy, is a simple method for achieving localization of single (or single groups of) fluorophores with nanometer accuracy in the xy plane. This protocol provides the steps necessary to run a control experiment, using DNA labeled with Cy3, to assess the efficacy of the FIONA setup and the level of attainable resolution. The Cy3-DNA is immobilized on a coverslip and imaged under deoxygenation conditions. It is important that the fluorophores remain photostable throughout the experiment. This requires an oxygen-scavenging system (e.g., glucose oxidase and catalase) in the medium.


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Constructing Sample Chambers for Fluorescence Imaging with One-Nanometer Accuracy (FIONA)
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