title: A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.
abstract: The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.
authors.affiliation: IMCF Biozentrum, Universität Basel , Klingelbergstrasse 50/70, 4056 Basel, Switzerland.
authors.forename: Wolf
authors.initials: W
authors.lastname: Heusermann
authors.affiliation: Life Imaging Services , Efringerstrasse 79, 4057 Basel, Switzerland.
authors.forename: Beat
authors.initials: B
authors.lastname: Ludin
authors.affiliation: School of Biological Sciences and School of Biomedical Sciences, University of Edinburgh , CH Waddington Building, Max Born Crescent, The King's Buildings, Edinburgh, EH9 3BF, United Kingdom.
authors.forename: Nhan T
authors.initials: NT
authors.lastname: Pham
authors.affiliation: School of Biological Sciences and School of Biomedical Sciences, University of Edinburgh , CH Waddington Building, Max Born Crescent, The King's Buildings, Edinburgh, EH9 3BF, United Kingdom.
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
authors.affiliation: Max Planck Institute of Biochemistry , Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weidemann
authors.affiliation: Bioseutica BV , Corso Elvezia 4, 6900 Lugano, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Hintersteiner
mesh_terms: D020650:Combinatorial Chemistry Techniques; D055808:Drug Discovery / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D019151:Peptide Library; D040081:Protein Array Analysis; D011485:Protein Binding
chemical_list: D019151:Peptide Library
keywords: high-throughput screening; one-bead one-compound libraries; spectral imaging; ultrafast screening