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Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

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Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam

Christophe Py¹, Danish Salim, Robert Monette

¹Institute for Microstructural Sciences, National Research Council of Canada, 1200 Montreal Rd, Ottawa, Ontario K1A0R6, Canada. christophe.py@nrc.ca

Biotechnology and Bioengineering

|March 11, 2011

Summary

Planar patch-clamp chips offer higher throughput for studying ion channels. This study introduces sieve chips and a dye-loading method to optimize cell-seal formation for improved electrophysiological measurements.

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Area of Science:

Electrophysiology
Cell Biology
Pharmacology

Background:

Patch-clamp electrophysiology is crucial for studying cellular electrical activity and drug effects on ion channels.
Planar patch-clamp chips are emerging as a high-throughput alternative to traditional glass-pipette methods.
Optimizing high-resistance seals between cells and chip apertures remains a challenge for accurate ion channel current measurement.

Purpose of the Study:

To develop and validate novel multiple-aperture (sieve) patch-clamp chips for enhanced throughput.
To introduce a reliable method for pre-screening cell-to-aperture seal quality.
To investigate cell-aperture interactions and seal formation in planar patch-clamp systems.

Main Methods:

Design and fabrication of multiple-aperture (sieve) patch-clamp chips.
Implementation of a dye loading protocol through chip apertures for seal quality assessment.
Evaluation of seal quality using fluorescence confocal microscopy and scanning electron microscopy (SEM).

Main Results:

Sieve chips facilitate rapid, statistically significant assessment of cell-to-aperture interactions.
The dye loading protocol effectively pre-screens the quality of cell-to-probe seals.
The study presents the first SEM image of a focused ion beam sectioned cell within a patch-clamp chip aperture.

Conclusions:

The developed sieve chips and dye-loading protocol significantly improve the optimization of cell-seal formation in planar patch-clamp assays.
This approach enhances the reliability and throughput of electrophysiological studies on ion channel proteins.
The findings provide a foundation for advancing high-throughput ion channel screening and drug discovery.