Simultaneous Measurement of Ca2+ and Cellular Dynamics: Combined Scanning Ion Conductance and Optical Microscopy to Study Contracting Cardiac Myocytes

Abstract

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell

“…1) based on a combination of SICM and SCM. The SICM used here was described (9,19,20) and includes feedback control based on distance modulation, a technique developed independently by us (9) and others (21). The sensitive SICM probe consists of a glass nanopipette filled with electrolyte with an Ag͞AgCl electrode plugged into it.…”

“…2A, in SSCM the cell is moved up and down in the z direction while scanning in the x and y directions, so its surface is always the same distance from the nanopipette (typically, 25-75 nm). A laser is passed up a high numerical aperture objective so that it is focused just at the tip of the nanopipette, and a pinhole is positioned at the image plane so that the confocal volume is just below the pipette, as described (9). Thus, a fluorescence image of the cell surface is obtained in a single scan, as well as a simultaneously captured image of the cell topography.…”

Characterization of Nanopipettes

“…Typical SICM experiments are performed in moderate 19 to high ionic strengths (>100 mM), 3,31,32,45,67,68 as are many nanopipette measurements. 14,15,18,19 Under these conditions, the diffuse double layer is expected to be compressed to a sub-nanometer scale, and therefore undetectable, level according to: 50…”

Multifunctional scanning ion conductance microscopy

“…Oscillation of the nanopipette position normal to the surface at an applied frequency, typically in the 100 Hz-1000 Hz range (figure 2e), results in an ionic current response at the same frequency that can be measured with a lock-in amplifier, which also provides the harmonic potential signal for the modulation of the z-piezoelectric positioner. The AC amplitude and/or phase response is much more stable [53][54][55][56] and more sensitive to nanopipette position near the surface. Using this approach, when the nanopipette is in bulk solution, typically more than one tip diameter away from the surface [4], the AC signal is small because there is little difference in the resistive state at the furthest and closest points of the probe-surface distance during the oscillation (figure 2e).…”