title: A PDMS-based biochip with integrated sub-micrometre position control for TIRF microscopy of the apical cell membrane.
abstract: A poly(dimethylsiloxane) (PDMS)-based biochip with an integrated pressure controlled positioning system with sub-micrometre precision was realized. The biochip was easy and cheap to manufacture and enabled positioning in a wet environment. It allowed the application of total internal reflection fluorescence (TIRF) microscopy at the dorsal cell membrane, which is not adhering to a support. Specifically, the chip enabled TIRF microscopy at the apical membrane of polarized epithelial cells. Thereby, the device allowed us for the first time to monitor individual fusion events of GPI-GFP bearing vesicles at the apical membrane in live Madin-Darby canine kidney II (MDCK II) cells. Moreover, a mapping of fusion sites became feasible and revealed that the whole apical membrane is fusion competent. In total, the biochip offers an all-in-one solution for apical TIRF microscopy and contributes a novel tool to study trafficking processes close to the apical plasma membrane in polarized epithelial cells.
authors.affiliation: Center for Advanced Bioanalysis GmbH, Scharitzerstrasse 6-8, 4020, Linz, Austria. roland.thuenauer@cbl.at
authors.forename: Roland
authors.initials: R
authors.lastname: Thuenauer
authors.forename: Kata
authors.initials: K
authors.lastname: Juhasz
authors.forename: Reinhard
authors.initials: R
authors.lastname: Mayr
authors.forename: Thomas
authors.initials: T
authors.lastname: Frühwirth
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Lipp
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Balogi
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D016764:Cell Polarity; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D004867:Equipment Design; D007091:Image Processing, Computer-Assisted; D056656:Lab-On-A-Chip Devices; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism; D046208:Microchip Analytical Procedures / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D011312:Pressure; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods; D022161:Transport Vesicles
chemical_list: D004129:Dimethylpolysiloxanes; D008565:Membrane Proteins; C013830:baysilon