A rapid-flow perfusion chamber for high-resolution microscopy.
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 approximately equal to 1 mLs-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.
Kaplan
D
D
Israel Institute for Biological Research, Ness-Ziona, Israel.
Bungay
P
P
Sullivan
J
J
Zimmerberg
J
J
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002459:Cell Fusion; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism; D004912:Erythrocytes; D005089:Exocytosis; D005347:Fibroblasts; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008561:Membrane Fusion; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods; D010063:Ovum / Q000378:metabolism; D012617:Sea Urchins
D002118:Calcium