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Abstract: Scanning ion conductance microscopy (SICM) is a scanning probe technique that utilizes the increase in access resistance that occurs if an electrolyte filled glass micro-pipette is approached towards a poorly conducting surface. Since an increase in resistance can be monitored before the physical contact between scanning probe tip and sample, this technique is particularly useful to investigate the topography of delicate samples such as living cells. SICM has shown its potential in various applications such as high resolution and long-time imaging of living cells or the determination of local changes in cellular volume. Furthermore, SICM has been combined with various techniques such as fluorescence microscopy or patch clamping to reveal localized information about proteins or protein functions. This review details the various advantages and pitfalls of SICM and provides an overview of the recent developments and applications of SICM in biological imaging. Furthermore, we show that in principle, a combination of SICM and ion selective micro-electrodes enables one to monitor the local ion activity surrounding a living cell.
Since the microscope has been invented in the 16th century, microscopes are used to study biological samples with a resolution beyond the limit of the human eye. Today, in general two different types of microscopes exist. The first and larger group comprises microscopes that operate in the far field. These microscopes use light or, in case of scanning or transmission electron microscopes, electrons to illuminate the sample and detect for example reflection, scattering or fluorescence. Since waves are used to illuminate the sample, these microscopes are subject to the diffraction limit that has been described by Ernst Abbe and that links the resolution of the microscope to the wavelength of the radiation used to illuminate the sample [1]. Nevertheless, recent developments in far-field microscopy allow to utilize certain physico-chemical properties of fluorescent molecules to circumvent Abbe's limit [2–6].
The second group of microscopes comprises the scanning probe microscopes. These microscopes use a tiny probe to measure a physical variable that depends on the distance between the probe and the sample surface. The first scanning probe microscope, the scanning tunneling microscope, was introduced in 1982 by Binnig, Rohrer and co-workers. It uses the tunneling current between a conducting probe and a conducting surface to determine positions of equal distance between tip and surface and thus to reconstruct the topography of conducting samples [7]. This technique, which was awarded with the Nobel Prize in 1986, requires the sample to be conductive and furthermore operates in vacuum, and hence can only image biological samples in an artificial environment after complex sample preparation. The second scanning probe technique developed by Binnig, Rohrer and their colleagues, the atomic force microscope (AFM) [8], utilizes the deflection of a soft cantilever with a sharp cone at its end that is dragged over the sample and hence senses its surface structure. Although the strengths of the AFM are the imaging of hard samples at even sub-atomic resolution [9], it had been quickly adapted to image biological samples [10–13]—the state of the art of AFM-imaging of biological samples has been reviewed recently [14]. Despite these successful recordings, the force which is applied by AFM to the sample cannot be neglected [15, 16] and hence imaging living cells without biasing still remains a challenging task.
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In 1989, Paul Hansma and colleagues invented the scanning ion conductance microscope (SICM) [17]. It monitors the ionic current through the tiny opening of an electrolyte filled glass micro- or nano-pipette. The current flow through the opening is hindered if the tip is in close proximity to a non-conducting surface. This relation between ionic current and tip-sample distance is utilized to determine the topography of the sample, either by calculating the distance between probe and sample from the current flowing through the tip opening, or, more often, by determining positions of equal resistance change which are used to reconstruct the sample topography.
The general assembly of a SICM is similar to electrophysiological setups, as shown in Figure 1A. A voltage is applied between two Ag/AgCl-electrodes, one of which is located in bulk electrolyte solution, the other one is inserted into a glass pipette containing the same electrolyte solution. The leakage current I
The mathematical descriptions of an ionic current through the aperture of a micro-pipette base on the description of the Scanning Electrochemical Microscope by Bard and colleagues [18, 19] that was adapted to SICM by the group of Harald Fuchs [20]. The different approaches and approximations to describe the current through a pipette depending on the shape of the pipette have been reviewed in detail recently [21], hence we focus on the description of the imaging process for a given pipette.
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Scanning electron micrographs of a scanning probe are shown in Figure 1B. The probe consists of a bulk region (top in Ba) and two tapering regions, the second one magnified in Figure 1Bb. The opening diameter of this pipette approximated one micrometer (Figure 1Bc, Bd). If combined with physiological bath solution, probes with this geometry have an access resistance in the range of 4 MOhm to 6 MOhm. A pipette like this one allows recordings with a resolution in the micrometer range. However, pipettes with a smaller opening diameter allow recordings with resolutions in the nanometer range. Using nano-pipettes with an opening diameter of 13 nm a spatial resolution in the range of 3 nm–6 nm has been observed [22]. However, the resolution of SICM still is a matter of debate, since in contrast theoretical considerations suggest a resolution of 3/2 inner pipette diameters [23].
As shown in Figure 2A, the circuit of a SICM can be described by a series of three resistors. The resistors r
Denote the resistances of the pipette and the bulk solution, respectively, and depend both on the conductance of the electrolyte as well as on the position and shape of the Ag/AgCl-electrodes (see [21] for review). The resistance r
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Through the aperture of the electrode depends, besides the shape of the tip, on the opening diameter and the distance d between the aperture and the sample surface [20, 23, 24].
Is a function of the position of the tip opening (and therefore of d) since it depends on the distance between tip opening and bath electrode. Furthermore, we neglect the voltage drop at the surfaces of the AgCl-electrodes, assuming they are constant and much smaller than the voltage drop at the pipette tip. Furthermore, we denote the reference resistance of the entire system at infinite tip-sample distance R(d = ∞) = R
A typical approach curve recorded with a micro-pipette as shown in Figure 1B is shown in Figure 2B. Note that since the initial distance d
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Between tip and sample is unknown, the deflection z of the piezo is recorded instead. The distance is the difference between the initial distance d
And D can be determined by numerically fitting this equation to the data. D is determined by the geometric properties of the electrode and the electrolyte. Basing on D, every distance D
T = D/ΔT. Since often recordings of approach curves end before the resistance is doubled, it might be more successful to directly fit
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Figure 3 shows an approach curve with Equation (3) fitted (red line) to the low-pass filtered data (blue crosses) of an approach curve, ΔT was chosen as 0.03 (indicated as relative threshold T = 1 + ΔT by the black line). For the given pipette, which was similar to the one in Figure 1B, D
Was 437.4 nm ± 16.0 nm (dashed black line). Note that here the approach curve was plotted versus the tip sample distance d, the piezo deflection is indicated by the gray axis at the top of the diagram. The resistance starts to increase approximately at a tip sample distance of 2.5 μm, that is approximately 2.5 inner diameters of the pipette. Nevertheless, a distinct signal that can be clearly distinguished from the remaining noise appears at approximately 1.5 μm (and hence 1.5 pipette diameters).
Since its invention, various ways to determine the
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