Potentiometric-scanning Ion Conductance Microscopy Research Articles

Quantitative Visualization of Nanoscale Ion Transport.

Understanding ion transport properties at various interfaces, especially at small length scales, is critical in advancing our knowledge of membrane materials and cell biology. Recently, we described potentiometric-scanning ion conductance microscopy (P-SICM) for ion-conductance measurement in polymer membranes and epithelial cell monolayers at discrete points in a sample. Here, we combine hopping mode techniques with P-SICM to allow simultaneous nanometer-scale conductance and topography mapping. First validated with standard synthetic membranes and then demonstrated in living epithelial cell monolayers under physiological conditions, this new method allows direct visualization of heterogeneous ion transport of biological samples for the first time. These advances provide a noncontact local probe, require no labeling, and present a new tool for quantifying intrinsic transport properties of a variety of biological samples.

Capturing Rare Conductance in Epithelia with Potentiometric-Scanning Ion Conductance Microscopy.

Tight junctions (TJs) are barrier forming structures of epithelia and can be described as tightly sealed intercellular spaces. Transport properties have been extensively studied for bicellular TJs (bTJs). Knowledge of the barrier functions of tricellular junctions (tTJs) are less well understood, due largely to a lack of proper techniques to locally measure discrete tTJ properties within a much larger area of epithelium. In this study, we use a nanoscale pipet to precisely locate tTJs within epithelia and measure the apparent local conductance of tTJs with a technique termed potentiometric scanning ion conductance microscopy (P-SICM). P-SICM shows the ability to differentiate transport through tTJs and bTJs, which was not possible with previous techniques and assays. We describe P-SICM investigations of both wild type and tricellulin overexpression Madin-Darby Canine Kidney (strain II, MDCKII) cells.

Alternating Current Potentiometric Scanning Ion Conductance Microscopy (AC-PSICM)

Studies of ion transport at small length scales inform the fundamental understanding of various biophysical processes. Here, we describe a new method, alternating current potentiometric scanning ion conductance microscopy (AC-PSICM), which measures ion transport through nanopores as a function of AC perturbations over a range of frequencies (5 Hz to 50 kHz). Phase and amplitude of local potential in the vicinity of nanopores in polymer membranes were captured with a nanopipet. Phase was found to be sensitive to local conductive pathways (nanopores in this case) and can be used to quantify single nanopore resistance. Investigation of phase approach curves and lateral phase distributions with single nanopore samples predicted four distinct frequency ranges for resolving heterogeneous conductive pathways within a sample, which were confirmed with line profile measurements of the phase response in samples with different sized nanopores. AC-PSICM is suitable for ion transport studies at the nanometer scale and can be used to access wide ranges of time scales. Phase mapping shows promise for visualization of heterogeneous transport pathways and could be used in future studies to examine conductance at cell and tissue interfaces.

Potentiometric-scanning ion conductance microscopy.

We detail the operation mechanism and instrumental limitations for potentiometric-scanning ion conductance microscopy (P-SICM). P-SICM makes use of a dual-barrel probe, where probe position is controlled by the current measured in one barrel and the potential is measured in a second barrel. Here we determine the interaction of these two barrels and resultant effects in quantitation of signals. Effects due to the size difference in pipet tip opening are examined and compared to model calculations. These results provide a basis for quantitation and image interpretation for P-SICM.

Scanning ion conductance microscopy measurement of paracellular channel conductance in tight junctions.

Elucidation of epithelial transport across transcellular or paracellular pathways promises to advance the present understanding of ion transport and enables regulation of cell junctions critical to the cell and molecular biology of the epithelium. Here, we demonstrate a new instrumental technique, potentiometric scanning ion conductance microscopy (P-SICM), that utilizes a nanoscale pipet to differentiate paracellular and transcellular transport processes at high spatial resolution. The technique is validated for well-defined polymer membranes and then employed to study wild type and claudin-deficient mutants of Madin-Darby Canine Kidney strain II (MDCKII) cells. Paracellular permeabilities conferred by claudin-2 are captured by P-SICM which demonstrates the utility to monitor apparent conductance at subcellular levels.