Monitoring cell movements and volume changes with pulse‐mode scanning ion conductance microscopy

Abstract

SummaryHere we describe the use of pulse-mode scanning ion conductance microscopy (SICM) to observe volume changes and cell membrane movements during the locomotion of cultured cells in the range of minutes to several hours. The microscope is based on the pulse-mode SICM previously developed for stable imaging of single cells in culture. Our instrument uses current pulses to control the distance between cell surface and electrode tip as well as a back-step mode to prevent contact of tip and membrane during lateral movements of the probe. We performed repeated scans of cell surfaces using feedbackcontrolled piezoactors to position the electrode. Using patchclamp-type electrode tips the height of cells could reproducibly be measured with a standard deviation of 50 nm. To quantify and separate changes in cell position and volume occurring between consecutive scans, a program was written to subtract images and calculate volume changes. Examples of repeated scans show that membrane movements in the range of 30 min to a few hours can be quantitatively monitored with a lateral resolution of 500 nm using difference images and that faster movements in the range of minutes can be recorded at defined cell sections using the line scan mode. Difference images indicate that volume changes can affect cell surfaces inhomogeneously, emphazising the role of the cytoskeleton in the stabilization of cell shape.

Scanning Ion Conductance Microscopy

“…Gitter et al [28], Mann et al [29] and Happel et al [30] employ a type of pointspectroscopy technique as it is know from other scanning probe methods such as atomic force microscopy [31]. Here the probe is approached to and then retracted from the sample surface by typically several micrometers in each data point of the scanning area.…”

“…One option is applying either short voltage or current pulses [29,30] instead of applying a constant voltage between the pipette and bath electrodes. This reduces unwanted effects caused by slowly changing dc potentials at the electrodes.…”

In-Phase Bias Modulation Mode of Scanning Ion Conductance Microscopy With Capacitance Compensation

“…ENBW is determined by the time constant T const and slope setting, and a simplified calculated formula is also concluded in the manual (pages [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. When the slope setting is constant at 24 dB/oct, the relationship between ENBW and T const is ENBW = 5/(64T const ).…”

Shear‐Force‐Controlled Scanning Ion Conductance Microscopy

“…One possible option is to employ some type of point-spectroscopy technique, as it is known from other scanning probe methods such as atomic force microscopy [29]. Here, the probe is approached to and then retracted from the sample surface by typically several micrometers for each data point of the scanning area [30][31][32]. Although the risk of the probe laterally colliding with a protrusion on the surface or it becoming trapped in a depression is reduced, this is achieved at the price of very low scanning speed.…”

“…Although the risk of the probe laterally colliding with a protrusion on the surface or it becoming trapped in a depression is reduced, this is achieved at the price of very low scanning speed. Unwanted effects caused by slowly changing direct current (DC) potentials at the electrodes can be reduced for instance by applying either short voltage or current pulses instead of applying a constant voltage between the nanopipette and bath electrodes [31,32].…”