title: Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns.
abstract: We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.
authors.affiliation: Biomolecular Photonics, Department of Physics, University of Bielefeld , Universitätsstraße 25, 33615 Bielefeld, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Hennig
authors.affiliation: Department of Biotechnology & Biophysics, Biozentrum, Julius Maximilians University Würzburg , Am Hubland, 97075 Würzburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: van de Linde
authors.affiliation: Biomolecular Photonics, Department of Physics, University of Bielefeld , Universitätsstraße 25, 33615 Bielefeld, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Bergmann
authors.affiliation: Biomolecular Photonics, Department of Physics, University of Bielefeld , Universitätsstraße 25, 33615 Bielefeld, Germany.|Department of Internal Medicine, NSF Center for Biophotonics, University of California, Davis , 2700 Stockton Boulevard, Suite 1400, Sacramento, California 95817, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.affiliation: Department of Biotechnology & Biophysics, Biozentrum, Julius Maximilians University Würzburg , Am Hubland, 97075 Würzburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D003543:Cysteamine / Q000737:chemistry; D004566:Electrodes; D005456:Fluorescent Dyes / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D011142:Polyvinyl Alcohol / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D055672:Static Electricity; D013269:Stochastic Processes
chemical_list: C569686:Alexa Fluor 647; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D003543:Cysteamine; D011142:Polyvinyl Alcohol; D005990:Glycerol
keywords: direct stochastic optical reconstruction microscopy (dSTORM); nanopipette; quantitative super-resolution microscopy; scanning ion conductance microscopy (SICM); single-molecule deposition