A rapid‐flow perfusion chamber for high‐resolution microscopy

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 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.

Analysis of Solution Exchange in Flow Chambers with Applications to Cell Membrane Permeability Measurement

“…However, some flow chambers designed by others had channel heights at least an order of magnitude larger than that used in the present study. 4,6,17,30 The inconsistencies in selection of channel dimensions may be due to confounding effects of fluid resistance (which increases with decreasing channel height) and the performance characteristics of the pump. Investigation of such effects was beyond the scope of the present study, which assumed that channel geometry and fluid velocity can be independently selected.…”

“…Other methods that have been used to estimate changes in perfusate composition at the channel wall include total internal reflection microfluorimetry 6 or pH measurement. 17 The report by Kaplan et al 17 is the only one that is confounded neither by measurement artifacts nor by inlet tubing dead volume, and as such, its data on solution exchange kinetics can be accepted as accurate. Therefore, the fact that the solution exchange rise times measured by Kaplan et al for Pe < 10 8 match within 10% the rise time predictions from our proposed empirical correlation (Eq.…”

Quantification of exocytosis kinetics by DIC image analysis of cortical lawns

“…Images were acquired at the focal plane of the coverslip surface during dilution of 1 ml of baseline intracellular medium (BIM) buffer (210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM PIPES, 50 mM CaCl 2 , 1 mMMgCl 2 , 1 mM EGTA, pH 6.7) [5, 6] containing 2 mM FITC and 10 % Mowiol-DABCO antifade by using an equal volume of this solution lacking FITC. Alternatively, a thin fluorescent protein layer was prepared from egg white protein labeled with FITC in carbonate buffer, pH 9.5 [11] to yield a fluorescent, denatured protein adhering firmly to the coverslip that is sensitive to pH changes. Gain or loss of FITC fluorescence intensity at the level of the coverslip was measured as above, either by initially equilibrating the protein at pH 8 and diluting it to pH 4 with addition of citrate buffer, or equilibrating at pH 4 and diluting to pH 8 with carbonate buffer.…”

Sea urchin egg preparations as systems for the study of calcium‐triggered exocytosis

“…A forward scattering microscope photometer is easily constructed using a darkfield condenser, a low numerical aperture objective and a photodetector. A stop‐flow system was designed to allow switching of solutions using turbulent rather than laminar flow (Kaplan et al 1996; Blank et al 1998 a ). Turbulent flow reduces the diffusion‐limited unstirred layer and allows a 95 % change in ionic concentration within μ0.3 s at the surface of the coverslip.…”

“…Turbulent flow reduces the diffusion‐limited unstirred layer and allows a 95 % change in ionic concentration within μ0.3 s at the surface of the coverslip. (Kaplan et al 1996). Faster rates of change in the free calcium concentration are achieved using a simple photolysis system employing a mercury arc lamp and solutions containing the photocleavable calcium chelator DM‐nitrophen (Vogel et al 1991; Shafi et al 1994).…”