Scanning Ion Conductance Microscopy Imaging Research Articles

Nanopipette Fabrication Guidelines for SICM Nanoscale Imaging.

Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.

Research on the Adaptive Sensitivity Scanning Method for Ion Conductance Microscopy with High Efficiency and Reliability.

Scanning ion conductance microscopy (SICM) is a type of in situ measurement technology for noncontact detection of samples in electrolytes with nanoscale resolution and has been used increasingly in biomedical and electrochemical fields in recent years. However, there is an inherent contradiction in the technique that makes SICM's sensitivity and accuracy difficult to balance. Higher sensitivity allows for faster probe speeds and higher scanning reliability but leads to lower accuracy, and vice versa. To resolve this problem, an adaptive sensitivity scanning method is proposed here that is designed to increase SICM's imaging efficiency without reducing its scanning reliability and accuracy. In the proposed scanning method, the sensitivity is automatically switched via the bias voltage based on the probe-sample distance. When the probe is located far away from the sample, the probe then predetects the sample position rapidly with high sensitivity. When the sample has been sensed in the high-sensitivity phase, the probe then detects the sample with low sensitivity. The basic theory and the feasibility of the alterable sensitivity detection strategy is also studied using the finite element method (FEM) and by performing experiments in this work. Finally, through testing of the standard silicon and polydimethylsiloxane (PDMS) samples, the proposed method is shown to increase SICM imaging efficiency significantly by up to 5 times relative to the conventional hopping mode without sacrificing the scanning accuracy and reliability.

Scanning ion conductance microscopy of isolated metaphase chromosomes in a liquid environment.

Scanning probe microscopy (SPM) uses a probing tip which scans over a sample surface for obtaining information on the sample surface characteristics. Among various types of SPM, atomic force microscopy (AFM) has been widely applied to imaging of biological samples including chromosomes. Scanning ion conductance microscopy (SICM) has been also introduced for visualizing the surface structure of biological samples because it can obtain "contact-free" topographic images in liquid conditions by detecting ion current flow through a pipette opening. However, we recently noticed that the consistent imaging of chromosomes is difficult by SICM. In this paper, the behaviors of the ion current on the sample surfaces were precisely investigated for obtaining SICM images of isolated muntjac metaphase chromosomes more consistently than at present. The present study revealed that application of positive potential to the pipette electrode was acceptable for obtaining the topographic image of chromosomes, while application of negative potential failed in imaging. The approach curves were then studied for analyzing the relationship between the ion current and the tip sample distance when the pipette is approaching chromosomes. The current-voltage (I-V) curve further provided us the accurate interpretation of the ion current behavior during chromosome imaging. These data were further compared with those for SICM imaging of HeLa cells. Our findings indicated that chromosomes are electrically charged and the net charge is strongly negative in normal Dulbecco's phosphate buffered saline. We finally showed that the ion concentration of the bath electrolyte is important for imaging chromosomes by SICM.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane.

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

High-Throughput Nanocapillary Filling Enabled by Microwave Radiation for Scanning Ion Conductance Microscopy Imaging.

Solid-state nanopores provide a highly sensitive tool for single-molecule sensing and probing nanofluidic effects in solutions. Glass nanopipettes are a cheap and robust type of solid-state nanopore produced from pulling glass capillaries with opening orifice diameters down to below tens of nanometers. Sub-50 nm nanocapillaries allow an unprecedented resolution for translocating single molecules or for scanning ion conductance microscopy imaging. Due to the small opening orifice diameters, such nanocapillaries are difficult to fill with solutions, compromising their advantages of low cost, availability, and experimental simplicity. We present a simple and cheap method to reliably fill nanocapillaries down to sub-10 nm diameters by microwave radiation heating. Using a large statistic of filled nanocapillaries, we determine the filling efficiency and physical principle of the filling process using sub-50 nm quartz nanocapillaries. Finally, we have used multiple nanocapillaries filled by our method for high-resolution scanning ion conductance microscopy imaging.

[Unlabeled imaging of primary cilia by scanning ion conductance microscopy].

Primary cilia are non-motile cilia consisting of a centriole-derived basal body and a microtubule-based axoneme. In recent years, the structure and function of primary cilia have been attracting attention due to the relation with the onset of ciliary disease. Scanning ion conductance microscopy (SICM) is a probe microscopy used to measure the topography and functions of living cells at nanoscale. Furthermore, the labelling procedure is not necessary for SICM measurement compare to fluorescence imaging. We compared the structures of primary cilia of human retinal pigment epithelial cell line (RPE-1 cells) and Madin-Darby canine kidney cell line (MDCK cells) at nanoscale by using SICM. In addition, high resolution SICM images have also succeeded in visualizing ciliary pockets that difficult to be fluorescently labeled.

A fast scanning ion conductance microscopy imaging method using compressive sensing and low-discrepancy sequences.

A scanning ion conductance microscope (SICM) is a multifunctional, high-resolution imaging technique whose non-contact nature makes it very suitable for imaging of biological samples such as living cells in a physiological environment. However, a drawback of hopping/backstep mode of SICM is its relatively slow imaging speed, which seriously restricts the study on the dynamic process of biological samples. This paper presents a new undersampled scanning method based on Compressed Sensing (CS-based scanning mode) theory to solve extended acquisition time issues in the hopping/backstep mode. Compressive sensing can break through the limit of the Nyquist sampling theorem and sample the original sparse/compressible signal at a rate lower than the Nyquist frequency. In the CS-based scanning mode, three sampling patterns, including the random sampling pattern and two kinds of sampling patterns produced by low-discrepancy sequences, were employed as the measurement locations to obtain the undersampled data with different undersampling ratios. Also TVAL3 (Total Variation Augmented Lagrangian ALternating-direction ALgorithm) was then utilized as a reconstruction algorithm to reconstruct the undersampled data. Compared with the nonuniform sampling points of random patterns at a low undersampling ratio, low-discrepancy sequences can produce a more uniform distribution point. Three types of samples with different complexity of topography were scanned by SICM using the conventional hopping/backstep mode and CS-based undersampled scanning mode. The comparisons of the imaging speed and quality with two scanning modes illustrate that the CS-based scanning mode can effectively speed up SICM imaging speed while not sacrificing the image quality. Also low-discrepancy sampling patterns can achieve a better reconstruction performance than that of the random sampling pattern under the same undersampling ratio.

Simultaneous scanning ion conductance and atomic force microscopy with a nanopore: Effect of the aperture edge on the ion current images

Scanning ion conductance microscopy (SICM) is a technique for high-resolution non-contact imaging, particularly powerful for live cell studies. Despite debates on its lateral resolution, consensus is that a probe presenting a tip with small opening aperture, large opening angle, and large outer-to-inner radius ratio will offer a SICM current signal more sensitive to tip-sample separation, ultimately impacting the image resolution. We report here the design of such a probe, integrating a nano-opening (<20 nm opening diameter) with increased outer-to-inner radius ratio and a wide opening angle through microfabrication and ion milling. The probe consists of a microfluidic atomic force microscopy (AFM) cantilever offered by the Fluid Force Microscope (FluidFM) technology, able to act as an SICM and AFM probe. Such a combination allows investigating the implications of the new probe geometry on the SICM imaging process by simultaneously recording currents and forces. We demonstrate through experiments on well-defined samples as well as corresponding simulations that by integrating a nanopore onto the FluidFM, nanoscale features could be successfully imaged, but the increased sensitivity of the probe current to sample distance comes with higher sensitivity to an inherent SICM wall artefact.

A fast imaging method of scanning ion conductance microscopy

Scanning ion conductance microscopy (SICM) has attracted considerable attention in the biological field as a noninvasive, high-resolution and non-force contact imaging technology. However, the development of improvement to the SICM imaging rate remains a great challenge for applications of rapid or dynamic imaging. In this paper, a fast SICM imaging method is proposed to improve the imaging efficiency via the design of a compressive sampling strategy and a reduction in the reconstruction time of sparse signals using the 2D normalized iterative hard thresholding (2D-NIHT) algorithm. The imaging performance of the method is validated by the simulation of recovery of a random synthetic image, and the superiority of the 2D-NIHT algorithm is also demonstrated by comparison of its reconstruction performance with that of other typical algorithms. The actual imaging performance of the method in SICM is also validated by the imaging of two biological samples, a virus and a living cell, and the results show that the method can duplicate the sample surface topography with high-definition and shorter imaging time. Our study offers a general imaging method for the applications of scanning probe microscopies to realize faster and higher-resolution imaging of biological samples.

Smart Scanning Ion-Conductance Microscopy Imaging Technique Using Horizontal Fast Scanning Method.

To solve extended acquisition time issues inherent in the conventional hopping-scanning mode of scanning ion-conductance microscopy (SICM), a new transverse-fast scanning mode (TFSM) is proposed. Because the transverse motion in SICM is not the detection direction and therefore presents no collision problem, it has the ability to move at high speed. In TSFM, the SICM probe gradually descends in the vertical/detection direction and rapidly scans in the transverse/nondetection direction. Further, the highest point that decides the hopping height of each scanning line can be quickly obtained. In conventional hopping mode, however, the hopping height is artificially set without a priori knowledge and is typically very large. Consequently, TFSM greatly improves the scanning speed of the SICM imaging system by effectively reducing the hopping height of each pixel. This study verifies the feasibility of this novel scanning method via theoretical analysis and experimental study, and compares the speed and quality of the scanning images obtained in the TFSM with that of the conventional hopping mode. The experimental results indicate that the TFSM method has a faster scanning speed than other SICM scanning methods while maintaining the quality of the images. Therefore, TFSM provides the possibility to quickly obtain high-resolution three-dimensional topographical images of extremely complex samples.

Nanoscale Imaging of Primary Cilia with Scanning Ion Conductance Microscopy.

Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.

A new scanning mode to improve scanning ion conductance microscopy imaging rate with pipette predicted movement

Scanning ion conductance microscopy (SICM) is a non-contact surface topography measurement technique that has been increasingly used for soft surfaces such as living biological samples. An approach-retract scanning (ARS) mode is widely used to avoid collision between the SICM probe (i.e., pipette) and an abrupt increase in sample profile. However, the redundant pipette trajectory in the ARS mode lengthens the scan time, thus reducing SICM efficiency and time resolution. To avoid this problem, a new scanning mode is discussed that adds horizontal movement at each measurement point to predict the upcoming sample topography via variation in ion current. The pipette then retracts in response to raised topography, while it raster scans flat or downhill topography. The feasibility was verified by finite element analysis and experimental tests on three kinds of soft samples: polydimethylsiloxane, mice cardiac fibroblasts, and breast cancer cells. The pixel detection frequency during imaging and the mean square error of the sample topography were compared for the two modes. The new scanning mode enhances the SICM imaging rate without loss of imaging quality or scanning stability, while it increases efficiency and time resolution. It thus has an improved performance for characterizing biological samples.

Dimensional comparison between amplitude-modulation atomic force microscopy and scanning ion conductance microscopy of biological samples

The range of scanning probe microscopy (SPM) applications for atomic force microscopy (AFM) is expanding in the biological sciences field, reflecting an increasing demand for tools that can improve our fundamental understanding of the physics behind biological systems. However, the complexity associated with applying SPM techniques in biomedical research hampers the full exploitation of its capabilities. Recently, the development of scanning ion conductance microscopy (SICM) has overcome these limitations and enabled contact-free, high resolution imaging of live biological specimens. In this work, we demonstrate the limitation of AFM for imaging biological samples in liquid due to artifacts arising from AFM tip–sample interaction, and how SICM imaging is able to overcome those limitations with contact-free scanning. We also demonstrate that SICM measurements, when compared to AFM, show better fit to the actual dimensions of the biological samples. Our results highlight the superiority of SICM imaging, enabling it to be widely adopted as a general and versatile research tool for biological studies in the nanoscale.

Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy.

Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

Nanoscale imaging of an unlabeled secretory protein in living cells using scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (∼500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.

Effect of Sample Slope on Image Formation in Scanning Ion Conductance Microscopy

Scanning ion conductance microscopy (SICM) is a scanning probe technique that allows investigating surfaces of complex, convoluted samples such as living cells with minimal impairment. This technique monitors the ionic current through the small opening of an electrolyte-filled micro- or nanopipet that is approached toward a sample, submerged in an electrolyte. The conductance drops in a strongly distance-dependent manner. For SICM imaging, the assumption is made that positions of equal conductance changes correspond to equal tip-sample distances and thus can be utilized to reconstruct the sample surface. Here, we examined this assumption by investigating experimental approach curves toward silicone droplets, as well as finite element modeling of the imaging process. We found that the assumption is strictly true only for perpendicular approaches toward a horizontal sample and otherwise overestimates the sample height by up to several pipet opening radii. We developed a method to correct this overestimation and applied it to correct images of fixed cellular structures and living entire cells.

Condenser-free contrast methods for transmitted-light microscopy.

Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared.

Efficient Imaging and Real-Time Display of Scanning Ion Conductance Microscopy Based on Block Compressive Sensing

Scanning Ion Conductance Microscopy (SICM) is one kind of Scanning Probe Microscopies (SPMs), and it is widely used in imaging soft samples for many distinctive advantages. However, the scanning speed of SICM is much slower than other SPMs. Compressive sensing (CS) could improve scanning speed tremendously by breaking through the Shannon sampling theorem, but it still requires too much time in image reconstruction. Block compressive sensing can be applied to SICM imaging to further reduce the reconstruction time of sparse signals, and it has another unique application that it can achieve the function of image real-time display in SICM imaging. In this article, a new method of dividing blocks and a new matrix arithmetic operation were proposed to build the block compressive sensing model, and several experiments were carried out to verify the superiority of block compressive sensing in reducing imaging time and real-time display in SICM imaging.

Bias Modulated Scanning Ion Conductance Microscopy

Nanopipets are versatile tools for nanoscience, particularly when used in scanning ion conductance microscopy (SICM) to determine, in a noncontact manner, the topography of a sample. We present a new method, applying an oscillating bias between a quasi-reference counter electrode (QRCE) in the SICM nanopipet probe and a second QRCE in the bulk solution, to generate a feedback signal to control the distance between the end of a nanopipet and a surface. Both the amplitude and phase of the oscillating ion current, induced by the oscillating bias and extracted using a phase-sensitive detector, are shown to be sensitive to the probe-surface distance and are used to provide stable feedback signals. The phase signal is particularly sensitive at high frequencies of the oscillating bias (up to 30 kHz herein). This development eliminates the need to physically oscillate the probe to generate an oscillating ion current feedback signal, as needed for conventional SICM modes. Moreover, bias modulation allows a feedback signal to be generated without any net ion current flow, ensuring that any polarization of the quasi reference counter electrodes, electro-osmotic effects, and perturbations of the supporting electrolyte composition are minimized. Both feedback signals, magnitude and phase, are analyzed through approach curve measurements to different surfaces at a range of distinct frequencies and via impedance measurements at different distances from a surface. The bias modulated response is readily understood via a simple equivalent circuit model. Bias modulated (BM)-SICM is compared to conventional SICM imaging through measurements of substrates with distinct topographical features and yields equivalent results. Finally, BM-SICM with both amplitude and phase feedback is used for topographical imaging of subtle etch features in a calcite crystal surface. The 2 modes yield similar results, but phase-detection opens up the prospect of faster imaging.

Method for Estimating the Tip Geometry of Scanning Ion Conductance Microscope Pipets

Scanning ion conductance microscopy (SICM) offers the ability to perform contact-free, high-resolution imaging of biological cells and tissues at physiological conditions. However, imaging resolution is highly dependent on the geometry of the SICM probe, which is generally not known. Small, high-resolution probes are too fine to image optically and, to date, geometry estimation has usually required electron microscopy (EM). This is time-consuming and prone to failure and cannot provide information about the crucial internal geometry of the probe. Here we demonstrate a new method for determining SICM tip geometry that overcomes the limitations of EM imaging. The method involves fitting an analytical model to current changes during quasi-controlled breakage of the pipet tip. The data can be routinely obtained using the SICM apparatus itself and our method thus opens the way for substantially better quantification in SICM imaging and measurement.