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Related Experiment Video

Updated: Sep 15, 2025

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

Published on: February 17, 2023

Optical Single-Channel Recording via Diffusional Confinement in Membrane Tethers.

Madeleine R Howell¹, Adam E Cohen^1,2

¹Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.

|July 15, 2025

Summary

This summary is machine-generated.

Researchers developed a novel membrane tether technique to observe individual ion transport events. This method enhances substrate concentration and observation time, enabling the study of low-conductance channels like CaV3.2.

Keywords:

fluorescent sensors live cell imaging membrane transport signal confinement single molecule

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Last Updated: Sep 15, 2025

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Area of Science:

Biophysics
Membrane Biology
Ion Channel Physiology

Background:

Single-channel electrophysiology is limited in studying ion channel gating when single-unit current is undetectable.
Probing membrane transport of substrates with low currents presents a significant technical challenge in biophysics.

Purpose of the Study:

To develop a novel method for observing and quantifying individual membrane transport events at the single-molecule level.
To overcome the limitations of detecting undetectable single-unit currents in membrane transport studies.

Main Methods:

Development of a membrane tether technique to isolate individual transmembrane proteins from live cells.
Utilizing fluorescent reporters within the tether to visualize and record individual transport events.
Imaging unitary Ca2+ transport events through the T-type Ca2+ channel (CaV3.2) in membrane tethers.

Main Results:

The membrane tether method achieved a ~1000-fold enhancement in substrate concentration and observation time compared to planar membranes.
Individual Ca2+ transport events were detected, corresponding to as little as ~0.4 fC of transported charge (6-13 Ca2+ ions).
Results were validated against ensemble electrophysiology and stochastic gating simulations.

Conclusions:

Tether-based single-channel recordings offer a powerful new approach to study the dynamics of membrane transport.
This technique enables the investigation of low-conductance ion channels and transporters previously inaccessible to single-channel analysis.
The method provides high sensitivity for detecting minimal ion fluxes across cell membranes.