PHYL3001 Lecture Notes - Lecture 2: Pipette, Reference Electrode, Electrophysiology
LECTURE TWO: Single Cell Electrophysiology Techniques
Methods in Single Cell Electrophysiology:
• In vitro (cell free) systems → artificial membranes
• In vivo systems → glass micropipette electrodes → sharp and patch
versions
Planar Lipid Bilayer:
• Thin membrane painted over small diameter hole
• Incorporate membrane proteins into bilayer
• Manipulate ionic composition on either side of membrane
• Measure transmembrane voltage via Ag/AgCl electrodes
Glass Micropipette Electrodes – Sharp Version:
• Technique
o Open tip <0.5um → access to intracellular solution inside cell and
avoids damage
o Filled with KCl solution
o Reference electrode stuck in solution (Ag/AgCl)
o Cell sits in bathing medium
o Electrode impales cell
o Second reference electrode in extracellular solution
o Potential difference measured
• Small tip size allows intracellular recording by piercing cell membrane
• Not all steady voltages are well-defined
• High resistance demands special electronics for recording devices
• High electrical resistance means lots of noise and interference
Diffusion Potentials:
• Because amount of charge movement is small, voltage is generated
without significant chances in concentration gradient
• Does not require complex intracellular machinery
• Voltage is not directly dependent on metabolic energy → dependent on
concentration gradient
• Voltage generated is proportional to concentration gradient →
relationship approximates but does not exactly follow Nernst prediction
• Vm depends on membrane permeability and concentration gradient
Voltage Clamp Method:
• Measuring ionic currents across cell membranes
• Current injected into cell causing step change in Vm
• When the recorded membrane potential deviates from the command/
voltage, further current is injected into the cell
• The current that needs to be passed through circuit to keep Vm constant =
total membrane current (Im)
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