A rapid-flow perfusion chamber for high-resolution microscopy |
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Authors: | D. KAPLAN,P. BUNGAY,J. SULLIVAN,& J. ZIMMERBERG |
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Affiliation: | Israel Institute for Biological Research, P.O.B. 19, Ness-Ziona 70450, Israel;, Biomedical Engineering &Instrumentation Program, NCRR, NIH, 13/3N17, Bethesda, MD 20892, U.S.A.;, Laboratory of Theoretical and Physical Biology, NICHD, NIH, 10/10D12 A, Bethesda, MD 20892, U.S.A. |
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Abstract: | Perfusion chambers employing laminar flow have dead volumes and unstirred layers which limit the minimum time required to effect a change in the local chemical environment of the sample. We have fabricated and tested a chamber capable of developing turbulent flow at reasonable flow rates of aqueous solutions. Transition to turbulence occurred at ≈1 mL s−1. To minimize dead space, a dual-exit cross-flow pattern was employed. The chamber was designed to mount on optical microscope stages for visual sample observation supplemented by a variety of techniques, such as fluorescence, light scattering and electrochemical monitoring. As indicated by fluorescence from a fluorescein-labelled protein film adherent to the chamber wall, local pH changes were produced within 200 ms. Use of the chamber is illustrated by measurements of stopped-flow kinetics in both calcium-triggered cortical granule exocytosis and influenza virus haemagglutinin-mediated cell–cell fusion. |
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Keywords: | Cell fusion exocytosis flow chamber kinetics stopped-flow |
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