Mode Note

Ion Conductance Microscopy (ICM)

Targeted Localized Stimulation and Monitoring of Cellular Activity
Non-Contact In-liquid Imaging and Nanoscale Electroscopy
The cell is the fundamental building block underlying all biological systems. Countless efforts have been made from various fields of science and technology to better understand this complex system. Now, we are opening a new chapter in the study of cells by introducing Ion Conductance Microscopy (ICM), a true technological breakthrough. Together with various optical microscopy techniques, this technological advancement will provide a unique and unprecedented opportunity in cell biology by enabling targeted localized stimulation and non-destructive monitoring of cellular activity heretofore inaccessible to other analytical techniques.

Atomic Force Microscopy (AFM) in Biology

Although nanometer-scale resolution can be achieved by electron microscopy (EM), samples must be frozen, fixed, dried, and processed prior to electron microscope imaging, and the morphological changes that result from such sample processing have always been a major concern for any EM studies. AFM, originally devised for material science, received some early attention for its potential biological imaging capabilities [1-3]. However, it is the ability to measure forces as indicated by the deflection of the AFM cantilever that has put it into prominence in surveying mechanical properties of biological sample surfaces [4]. Along the way, various imaging techniques [5, 6] were developed to study biological structures and functions including inert material-mounted stylus for imaging live cells submerged in physiological buffer solutions [7] as well as the AFM in combination with optical spectroscopy [8]. Currently, AFM technology is rapidly adding new capabilities to detect various physical properties and manipulate biological entities at the nanoscale.

Ion Conductance Microscopy

Independently, a different SPM technology was developed by Hansma et al. in 1989, offering a remarkable non-contact liquid imaging capability [9]. In Ion Conductance microscopy (ICM or SICM for the acronym of scanning), a glass nanopipette (See Figure 1) filled with an electrolyte senses ion current to feedback its position relative to samples completely immersed in a liquid buffer. Since the tip-sample distance is maintained by keeping the ionic current constant instead of applying a physical force to the sample, it is an ideal tool to obtain a stable image of soft and sticky biological samples [10, 11].

AFM Probe

ICM Probe: Nanopipette

Fig. 1 Unlike AFM where micro-machined cantilever is used as a probe, ICM utilizes a pipette probe made of glass or quartz whose inner diameters range 80~100 nm for glass and 30 - 50 nm for quartz respectively.

Similar to Scanning Tunneling Microscopy in ambient air, the ICM operates in liquid without physical contact with the sample. One electrode is placed inside of the pipette, while another is located in a bath solution (See Figure 2). When an external bias is applied between these two electrodes, a current flow is detected through conducting ions. In completing the overall electrical circuit, one needs to account for two electrical resistances at the channel assuming that the resistance of the bath solution is negligible. The first electrical resistance emanates from the frustum shape of the pipette while the second results from the distance between the pipette and the sample surface. When the pipette is far from the surface, the latter electrical resistance diminishes, reaching a saturated current because the resistance due to the tip shape is almost constant during the measurement (See Figure 3a). As the pipette gets closer to the sample however, the volume of the conductive ion channel between the probe and the sample becomes smaller (See Figure 3a), resulting in a rapid decrease of the ionic current, which is in turn used as a reference feedback signal (See Figure 3b). One can also apply an AC modulation to the technique in order to achieve a more stable operation [10] during measurement.

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Although ICM was developed many years ago, it has not been widely used during the last decade due to the instrumentation complexity and the subsequent operational instability, in particular the large Z-bandwidth requirement for proper Z-servo feedback, a key bottleneck overcome by the XE-Bio.

Live Cell Membrane Imaging with SICM

The cell membrane is probably the most important component of a cell. Most of cellular activities are mediated via the membrane, the only cellular structure found in all types of cells in living organisms. However, it is extremely difficult to monitor a live cell membrane at the nanometer scale. In particular, the transparency of the membrane makes it virtually impossible to observe with optical microscopy. Figure 4 shows SICM images of live COS-1 cells, which are transformed from CV-1 fibroblast with simian virus 40 (SV40) from the normal kidney adult African green monkey. The cells were live and stable during the entire duration of ICM imaging, showing no signs of physical deterioration. The fibroblast line adheres to glass and plastic in culture and is generally utilized as a transfection host. The yellow arrows in Figs. 4(a) and 4(b) show how fibroblasts behave when two growing cells membranes collide. Often, two neighboring cells exhibit different levels of cilia activity as shown in Figs. 4(c) and 4(d). A Higher density of cilia structure can be observed in the fibroblast A compared to B and such different densities are even more evident in the phase image of Figure 4(d). Such structural differences are almost impossible to observe with an optical microscopy or traditional AFM. ICM topography image of mouse lung cell in Figure 5(a) nicely shows the details of a living cell whose measured image is completely different in dead and dried cell. Furthermore, ICM current error image in Figure 5(b) displays the cell traction mark on the bottom after cell contraction of live mouse muscle cell (C2C12). Consecutive ICM images in Figure 6 show microvilli on the cell surface and the sustained structures of the cell membrane during the zooming in process.

Current Amp

ICM Probe: Nanopipette Ag/AgCl electrode

Control System Z Mod

Live Cells in the buffer solution

Fig. 2 In ICM, a current flow between two electrodes is detected through conducting ions in the solution. As the pipette gets closer to the sample surface, the volume of conductive ion channel between two electrodes becomes smaller, rapidly decreasing the ionic current.

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Fig.3 Schematic diagram of SICM Operation

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Fig. 4 SICM images of live COS-1 cell: (a) and (c) are SICM images whose scan size are 30 um and 40 um, respectively. (b) and (d) are corresponding phase images.

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Fig. 5 ICM top ography of live mouse lung cell (a)* and ICM current error images of live mouse muscle cell (C2C12) (b) * By courtesy of Prof D. Anselmetti and his group at the University of Bielefeld, Germany

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Fig. 6 SICM of liver cell

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Fig. 7 Targeted localized stimulation can be accomplished by applying a controlled pressure through the pipette hole whose glass surface can be functionalized per customers need.

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Targeted Localized Stimulation and Monitoring of Cellular Activity

Using a fluid filled pipette for ICM instead of a silicon cantilever for AFM opens pathways for new analytical possibilities. Ideal for imaging soft biological samples in liquid, such as living cells, ICM can be easily adapted to a host of qualitative and quantitative biochemical stimulation on single cells and cell motility studies, whose applications include targeted localized stimulation and monitoring (See Figure 7), and cellular drug delivery. In targeted localized stimulation, one induces a cell movement by applying a localized pressure via the pipette hole and monitors the subsequent responses [11]. Furthermore, the functional capability of the ICM can be extended to the study of live cell dynamics in response to targeted chemical or drug stimulation, achieving precisely controlled electrophysiology at the nanoscale. The field of single cell research is now accessible to everyone who is interested in, and this powerful ICM technique will revolutionize the field of Cell Biology including drug delivery research.

New Bio-Convergence Solution, XE-Bio

Park Systems introduced the XE-Bio, an enabling bio solution for biomedical and life science, uniquely combining non-contact Atomic Force Microscopy (AFM) and Ion Conductance Microscopy (ICM). The modular design of the XE-Bio allows easy exchange between noncontact AFM and ICM. Designed for non-invasive in-liquid operation, the combined imaging capability of AFM, ICM, and inverted optical microscopy makes the XE-Bio ideal for imaging biological samples in dynamic conditions such as living cells in liquid. Moreover, ICM can be further adapted to enable a host of powerful applications in nanoscale electrophysiology.

Reference
1. Alexander et al., J. Appi. Phys. 65: 164-167 (1989) 2. Rugard and Hansma, Physics Today 43: 23-30 (1990) 3. Jena and Cho, Methods in Cell Biology 68:33-50 (2002) 4. Weisenhorn, et al. Nanotechnology 4:106113 (1993) 5. Hrber et al., Scan.Microsc.6:919 ( 1992) 6. Spudich and Braunstein, Proc. Natl. Acad. Sci. USA. 92: 6976-6980 (1995) 7. Micic et al., Colloids Surf B Biointerfaces 34(4): 205-12 (2004) 8. Danker and Oberleithner, 439(6): 671-81 (2000) 9. Hansma et al., Science 243(4891): 6413 (1989) 10. Pastre et al., Ultramicroscopy, 90(1):139, (2001) 11. Korchev et al., Nature Cell Biology 2: 616-9 (2000)

Specifications
ICM Head (Ion Conductance Microscopy) Faraday cage Low noise current amplifier Pipette holder including Ag/AgCl electrodes Operating Mode: ICM (DC and AC mode available) Bias range: -10 - + 10 V Current range: 103 - 1011 V/A Lateral resolution: Pipette size limited Z scan range: 25 m Pipette -Outer diameter: 1.0 mm -Inner diameter: 30 - 50 nm (quartz), 80 - 100 nm (glass)

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