Realizing the Biological and Biomedical Potential of Nanoscale Imaging Using a Pipette Probe

Andrew I Shevchuk; Pavel Novak; Yasufumi Takahashi; Richard Clarke; Michele Miragoli; Babak Babakinejad; Julia Gorelik; Yuri E Korchev; David Klenerman


Nanomedicine. 2011;6(3):565-575. 

In This Article

Abstract and Introduction


Cells naturally operate on the nanoscale level, with molecules combining together to form complex molecular machines, which can work together to enable normal cell function or go wrong as in the case of many diseases. Visualizing these key processes on the nanoscale has been difficult and two main approaches have been used to date; nanometer resolution imaging of fixed cells using electron microscopy, or imaging live cells using optical or fluorescence microscopy, with a resolution of a few hundred nanometers. Scanning probe microscopy has the potential to allow live cells to be imaged at nanoscale resolution and a noncontact method based on the use of a nanopipette probe has been developed over the last 10 years that allows both topographic and functional imaging. The rapid progress in this area of research over the last 4 years is reviewed in this article, which shows that imaging of complex cellular structures and tissues is now possible and that these methods are now sufficiently mature to provide new insights into important diseases.


In the middle of the 1980s, when scanning probe microscopy was first developed, one of the aims was to use this method to image the topography of live cells with a resolution comparable to an electron microscope. However, despite recent advances in the biological applications of atomic force microscopy (AFM), its resolution on living eukaryotic cells remains comparable to optical imaging.[1,2] For this reason, fluorescence microscopy remains the most widely used method for live cell imaging. Although fluorescence imaging provides molecular specificity, it cannot visualize important topological changes taking place on surfaces of live cells at the nanoscale. Scanning ion conductance microscopy (SICM), invented by Paul Hansma in 1989, has been successfully adopted for topographical imaging of live biological cells under physiological buffer.[3,4] Nevertheless, the lack of sideways sensitivity of the SICM probe, which is a glass micropipette or nanopipette, has limited the application of SICM to the imaging of relatively flat surfaces since its invention. In fact, this is a fundamental problem for all scanning probe microscopes, since the sidewall of the probe can touch the sample before the tip of the probe has sensed the presence of this object (Figure 1A), resulting in sample and/or probe damage (Figure 1C). The introduction of the distance-modulated feedback protocol, which we reviewed in a previous technical report 4 years ago, improved the SICM feedback stability and allowed the imaging of more complex structures, but did not solve the fundamental problem of sideways sensitivity.[5,6] As a result, the scanning pipette always collided with obstacles taller than the modulation amplitude. However, the recent introduction of hopping probe ion conductance microscopy (HPICM) by Novak et al.[7] has now made it possible to image the highly convoluted surfaces of live biological cells at a resolution better than 20 nm, as shown in Figure 1B & 1D. HPICM has some conceptual similarities to the back step mode by Happel et al.[8] Manipulation of cells at the submicron scale using pulse mode SICM has also recently been reported.[9] However, despite the conceptual simplicity of withdrawing the probe to a certain height before positioning it to a new location to be measured, this protocol results in a prohibitively slow imaging rate.[8,10] For example, an image of a size of 256 by 256 pixels, with a probe withdrawn by 5 µm at every pixel, would take more than 2 h to acquire. This is currently due to the response time of the piezo actuator, which is in the milliseconds range. To address this issue Novak et al. developed adaptive scanning, where the sample is scanned at variable resolution, depending on how rough or smooth its surface is, see Figure 2. Adaptive scanning actually reduces the scan time significantly, opening up the possibility of imaging topographic changes on the cell surface in real time. This key advance of the combination of adaptive SICM and HPICM is described in more detail below.

Figure 1.

Comparison of continuous/distance-modulated scanning ion conductance microscopy and hopping probe ion conductance microscopy operation. (A) A scanning nanopipette probe operating in continuous scan mode collides with a large spherical object. (B) Hopping mode, used in hopping probe ion conductance microscopy, avoids collision by withdrawing the same pipette to a position well above the sample. (C & D) An example of improved imaging using hopping probe ion conductance microscopy. Topographical images of the same fixed vertically protruding mechanosensitive stereocilia of auditory hair cells, obtained first with continuous raster scan mode (C) and then with hopping mode (D), using the same nanopipette. Both images took 30 min to acquire.

Figure 2.

Principle of adaptive resolution. (A) For adaptive resolution hopping probe ion conductance microscopy, the scan area is first divided into equally sized squares (bottom). Before imaging each square, the roughness of the sample in this square was estimated using the height at its corners highlighted in red (middle). Very rough squares were imaged at high resolution (top right) and smoother squares were imaged at low resolution (top left). (B) As an example, a 3D topographical rendered image of a hippocampal pyramidal neuron was acquired using this adaptive scanning algorithm (bottom). The resolution that was used for imaging this neuron (high resolution in dark green, low resolution in light green) is shown above.


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