Scanning Ion Conductance Microscopy Research Articles

Membrane fluctuations of human red blood cells investigated by the current signal noise in scanning ion conductance microscopy

Human red blood cells show submicron membrane fluctuations (CMFs) that have been mainly studied with optical microscopies. Although the functional role of this phenomenon is still uncertain, the amplitude of membrane fluctuations is considered as an indicator of mechanical resilience to the stress encountered in the capillary beds. We investigate here the membrane fluctuations in red blood cells using the scanning ion conductance microscopy (SICM), a scanning probe technique that avoids the probe-sample contact. The ion current noise was recorded at a fixed distance from the cell and converted in terms of membrane fluctuation amplitude using as a converting factor the slope of the current-distance curve. We found that CMF amplitude was irreversibly reduced by membrane cross-link. Both whole cell and local increase of membrane tension induced a reduction of CMF amplitude. As for the biochemical regulation of membrane dynamics, we observed that the activation of the noradrenergic transduction pathway, via β-receptors, increased the CMF amplitude. We conclude that the CMFs recorded by SICM and those optically recorded on red blood cells share the main features. In addition SICM provides high spatial and temporal resolution as well as the possibility to apply through the glass pipette acting as probe chemical or physical stimuli to the membrane area where the CMFs are recorded.

Simultaneous Scanning Ion Conductance and Electrochemical Microscopy in Lithium‐Ion Battery Research

AbstractInvited for this issue's Front Cover is the group of Prof. F.‐M. Matysik. The cover picture shows a typical situation in the life of a scientist focusing on scanning probe techniques. A decision needs to be made, which technique to employ. But what if more than one technique could be employed. In the context of lithium‐ion battery research, several scanning probe microscopies are currently utilized. Scanning ion conductance microscopy and scanning electrochemical microscopy were simultaneously applied in the investigation of a commercial graphite electrode. Read the full text of the Research Article at 10.1002/celc.202300577.

Single-cell nanobiopsy enables multigenerational longitudinal transcriptomics of cancer cells.

Single-cell RNA sequencing has revolutionized our understanding of cellular heterogeneity, but routine methods require cell lysis and fail to probe the dynamic trajectories responsible for cellular state transitions, which can only be inferred. Here, we present a nanobiopsy platform that enables the injection of exogenous molecules and multigenerational longitudinal cytoplasmic sampling from a single cell and its progeny. The technique is based on scanning ion conductance microscopy (SICM) and, as a proof of concept, was applied to longitudinally profile the transcriptome of single glioblastoma (GBM) brain tumor cells in vitro over 72 hours. The GBM cells were biopsied before and after exposure to chemotherapy and radiotherapy, and our results suggest that treatment either induces or selects for more transcriptionally stable cells. We envision the nanobiopsy will contribute to transforming standard single-cell transcriptomics from a static analysis into a dynamic assay.

Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Effect of LDL Extracted from Human Plasma on Membrane Stiffness in Living Endothelial Cells and Macrophages via Scanning Ion Conductance Microscopy.

Mechanical properties of living cells play a crucial role in a wide range of biological functions and pathologies, including atherosclerosis. We used low-stress Scanning Ion-Conductance Microscopy (SICM) correlated with confocal imaging and demonstrated the topographical changes and mechanical properties alterations in EA.hy926 and THP-1 exposed to LDL extracted from CVD patients' blood samples. We show that the cells stiffened in the presence of LDL, which also triggered caveolae formation. Endothelial cells accumulated less cholesterol in the form of lipid droplets in comparison to THP-1 cells based on fluorescence intensity data and biochemical analysis; however, the effect on Young's modulus is higher. The cell stiffness is closely connected to the distribution of lipid droplets along the z-axis. In conclusion, we show that the sensitivity of endothelial cells to LDL is higher compared to that of THP-1, triggering changes in the cytoskeleton and membrane stiffness which may result in the increased permeability of the intima layer due to loss of intercellular connections and adhesion.

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.

Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

500-kHz Bandwidth Feedback for Scanning Ion Conductance Microscopy With Nanoresolution

Scanning ion conductance microscopy (SICM) has been developing rapidly and has been a versatile tool for nanoscale imaging. However, the imaging quality and scanning speed of SICM have been significant restriction factors for its various applications. One of the main reasons is the mutual restriction of bandwidth and noise of the ion current feedback. In this paper, a low-noise two-stage transimpedance amplifier (TIA) with 500 kHz bandwidth was proposed and a low-capacitance pipette was combined to depress the high-frequency ion current noise of SICM's feedback circuit. The effect of pipette capacitance, which is a major cuase of high-frequency noise, was significantly reduced by covering a thin polydimethylsiloxane (PDMS) film on the outer wall of the pipette. Finite element method (FEM) was employed for the first time to simulate the pipette capacitance model. Impedance spectroscopy experiments verified that this method can greatly reduce the pipette capacitance (about 0.7 times). Through the comparative analysis of ion current noise, as well as the scanning experiments of standard silicon material sample and PMDS sample, it was demonstrated that the proposed method has a significant effect in reducing the high frequency noise of ion current measurement. Therefore, our method can provide an avenue to realize high-speed imaging with high positioning accuracy.

Cardiac-Specific Deletion of Scn8a Mitigates Dravet Syndrome-Associated Sudden Death in Adults.

Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear. In this study the authors sought to provide evidence implicating remodeling of Na+ - and Ca2+ -handling machinery, including NaV 1.6 and Na+/Ca2+exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias. The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS. DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death. These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts servingas a substrate for Ca2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention ofSUDEPin adultDS subjects.

Simultaneous Scanning Ion Conductance and Electrochemical Microscopy in Lithium‐Ion Battery Research

AbstractDeeper understanding of processes involved in operation of lithium‐ion batteries (LIBs) is necessary to further optimize them for future applications. Extensive research was conducted on the formation of a solid electrolyte interphase (SEI) on negative battery electrodes, still leaving several questions unanswered. Scanning probe microscopies (SPMs) enable in situ and operando investigations and have the potential to explain some phenomena. Scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) could be employed in LIB studies. A novel SICM method based on the redox couple ferrocene/ferrocenium is introduced for applications in carbonate solvents widely used in LIBs. Proof of concept measurements were conducted with a micro milled copper circuit board as model substrate. Furthermore, the proposed SICM approach was hyphenated with feedback mode SECM resulting in the simultaneous mapping of morphology and electrochemical activity. A flexible dual‐probe arrangement was developed enabling usage of both SPM techniques at the same time, and furthermore, an easy replacement of both individual probes if needed. The setup was applied in the characterisation of commercial graphite electrodes for LIBs before and after conducting a pre‐charging protocol. Changes in electrochemical activity and topography of the graphite electrode were resolved in simultaneously generated SECM/SICM recordings.

Scanning ion conductance microscopy revealed cisplatin-induced morphological changes related to apoptosis in single adenocarcinoma cells.

The studies of drug-induced apoptosis play a vital role in the identification of potential drugs that could treat diseases such as cancer. Alterations in the native morphology of cancer cells following treatment with anticancer drugs serve as one of the indicators that reveal drug efficacy. Various techniques such as optical microscopy, electron microscopy (EM), and atomic force microscopy (AFM) have been used to map the three dimensional (3D) morphological changes in cells induced with drugs. However, caution should be exercised when interpreting morphological data from techniques that might alter the native morphology of cells, caused by phototoxicity, electron beam invasiveness, intrusive sample preparation, and cell membrane deformation. Herein, we have used scanning ion conductance microscopy (SICM) to study the 3D morphology and roughness of A549 adenocarcinoma cells under physiological conditions before and after cisplatin induced apoptosis, where we observed an increase in height, overall shrinkage of the cells, and irregular features form on the cell membrane. Tracking the morphology of the same single A549 cells exposed to cisplatin unveiled heterogeneity in response to the drug, formation of membrane blebs, and an increase in membrane roughness. We have also demonstrated the use of SICM for studying the effect of cisplatin on the dynamic changes in the volume of A549 cells over days. SICM is demonstrated as a technique for studying the effect of drug induced apoptosis in the same cells over time, and for multiple different single cells.

Applications of scanning probe microscopy in neuroscience research

Abstract Scanning probe microscopy techniques allow for label-free high-resolution imaging of cells, tissues, and biomolecules in physiologically relevant conditions. These techniques include atomic force microscopy (AFM), atomic force spectroscopy, and Kelvin probe force microscopy, which enable high resolution imaging, nanomanipulation and measurement of the mechanoelastic properties of neuronal cells, as well as scanning ion conductance microscopy, which combines electrophysiology and imaging in living cells. The combination of scanning probe techniques with optical spectroscopy, such as with AFM-IR and tip-enhanced Raman spectroscopy, allows for the measurement of topographical maps along with chemical identity, enabled by spectroscopy. In this work, we review applications of these techniques to neuroscience research, where they have been used to study the morphology and mechanoelastic properties of neuronal cells and brain tissues, and to study changes in these as a result of chemical or physical stimuli. Cellular membrane models are widely used to investigate the interaction of the neuronal cell membrane with proteins associated with various neurological disorders, where scanning probe microscopy and associated techniques provide significant improvement in the understanding of these processes on a cellular and molecular level.

Rapid scanning method for SICM based on autoencoder network

Scanning Ion Conductance Microscopy (SICM) enables non-destructive imaging of living cells, which makes it highly valuable in life sciences, medicine, pharmacology, and many other fields. However, because of the uncertainty retrace height of SICM hopping mode, the time resolution of SICM is relatively low, which makes the device fail to meet the demands of dynamic scanning. To address above issues, we propose a fast-scanning method for SICM based on an autoencoder network. Firstly, we cut under-sampled images into small image lists. Secondly, we feed them into a self-constructed primitive-autoencoder super-resolution network to compute high-resolution images. Finally, the inferred scanning path is determined using the computed images to reconstruct the real high-resolution scanning path. The results demonstrate that the proposed network can reconstruct higher-resolution images in various super-resolution tasks of low-resolution scanned images. Compared to existing traditional interpolation methods, the average peak signal-to-noise ratio improvement is greater than 7.5823 dB, and the average structural similarity index improvement is greater than 0.2372. At the same time, using the proposed method for high-resolution image scanning leads to a 156.25% speed improvement compared to traditional methods. It opens up possibilities for achieving high-time resolution imaging of dynamic samples in SICM and further promotes the widespread application of SICM in the future.

The target region focused imaging method for scanning ion conductance microscopy

Scanning ion conductance microscopy (SICM) has developed rapidly and has wide applications in biomedicine, single-cell science and other fields. SICM scanning speed is limited by the conventional raster-type scanning method, which spends most of time on imaging the substrate and does not focus enough on the target area. In order to solve this problem, a target region focused (TRF) method is proposed, which can effectively avoid the scanning of unnecessary substrate areas and enables SICM to image the target area only to achieve high-speed and effective local scanning. TRF method and conventional hopping mode scanning method are compared in the experiments using breast cancer cells and rat basophilic leukemia cells as experimental materials. It was demonstrated that our method can reduce the scanning time for a single sample image significantly without losing scanning information or compromising the quality of imaging. The TRF method developed in this paper can provide an efficient and fast scanning strategy for improving the imaging performance of SICM systems, which can be applied to the dynamic features of cell samples in the fields of biology and pharmacology analysis.

Latest Advances in Scanning Ion-Conductance Microscopy and Nanopipette Systems for Single-Cell Analysis under Serial Conditions

Latest Advances in Scanning Ion-Conductance Microscopy and Nanopipette Systems for Single-Cell Analysis under Serial Conditions

Visualization of the membrane surface and cytoskeleton of oligodendrocyte progenitor cell growth cones using a combination of scanning ion conductance and four times expansion microscopy.

Growth cones of oligodendrocyte progenitor cells (OPCs) are challenging to investigate with conventional light microscopy due to their small size. Especially substructures such as filopodia, lamellipodia and their underlying cytoskeleton are difficult to resolve with diffraction limited microscopy. Light microscopy techniques, which surpass the diffraction limit such as stimulated emission depletion microscopy, often require expensive setups and specially trained personnel rendering them inaccessible to smaller research groups. Lately, the invention of expansion microscopy (ExM) has enabled super-resolution imaging with any light microscope without the need for additional equipment. Apart from the necessary resolution, investigating OPC growth cones comes with another challenge: Imaging the topography of membranes, especially label- and contact-free, is only possible with very few microscopy techniques one of them being scanning ion conductance microscopy (SICM). We here present a new imaging workflow combining SICM and ExM, which enables the visualization of OPC growth cone nanostructures. We correlated SICM recordings and ExM images of OPC growth cones captured with a conventional widefield microscope. This enabled the visualization of the growth cones' membrane topography as well as their underlying actin and tubulin cytoskeleton.

Opto-SICM framework combines optogenetics with scanning ion conductance microscopy for probing cell-to-cell contacts

We present a novel framework, Opto-SICM, for studies of cellular interactions in live cells with high spatiotemporal resolution. The approach combines scanning ion conductance microscopy, SICM, and cell-type-specific optogenetic interrogation. Light-excitable cardiac fibroblasts (FB) and myofibroblasts (myoFB) were plated together with non-modified cardiomyocytes (CM) and then paced with periodic illumination. Opto-SICM reveals the extent of FB/myoFB-CM cell-cell contacts and the dynamic changes over time not visible by optical microscopy. FB-CM pairs have lower gap junctional expression of connexin-43 and higher contact dynamism compared to myoFB-CM pairs. The responsiveness of CM to pacing via FB/myoFB depends on the dynamics of the contact but not on the area. The non-responding pairs have higher net cell-cell movement at the contact. These findings are relevant to cardiac disease states, where adverse remodeling leads to abnormal electrical excitation of CM. The Opto-SICM framework can be deployed to offer new insights on cellular and subcellular interactions in various cell types, in real-time.

Scanning Ion-Conductance Microscopy for Studying β-Amyloid Aggregate Formation on Living Cell Surfaces.

β-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of β-amyloid (Aβ) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aβ aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aβ leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aβ aggregates on living cell surfaces.

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate.

Mechanical properties of neuronal cells have a key role for growth, generation of traction forces, adhesion, migration, etc. Mechanical properties are regulated by chemical signaling, neurotransmitters, and neuronal ion exchange. Disturbance of chemical signaling is accompanied by several diseases such as ischemia, trauma, and neurodegenerative diseases. It is known that the disturbance of chemical signaling, like that caused by glutamate excitotoxicity, leads to the structural reorganization of the cytoskeleton of neuronal cells and the deviation of native mechanical properties. Thus, to investigate the mechanical properties of living neuronal cells in the presence of glutamate, it is crucial to use noncontact and low-stress methods, which are the advantages of scanning ion-conductance microscopy (SICM). Moreover, a nanopipette may be used for the local delivery of small molecules as well as for a probe. In this work, SICM was used as an advanced technique for the simultaneous local delivery of glutamate and investigation of living neuronal cell morphology and mechanical behavior caused by an excitotoxic effect of glutamate.

Measuring Melanoma Nanomechanical Properties in Relation to Metastatic Ability and Anti-Cancer Drug Treatment Using Scanning Ion Conductance Microscopy.

A cell's mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, reliable cancer marker. For example, it has been widely published that cancer cells show a lower Young's modulus than their non-cancerous counterparts. Furthermore, the effect of anti-cancer drugs on cellular mechanics may offer a new insight into secondary mechanisms of action and drug efficiency. Scanning ion conductance microscopy (SICM) offers a nanoscale resolution, non-contact method of nanomechanical data acquisition. In this study, we used SICM to measure the nanomechanical properties of melanoma cell lines from different stages with increasing metastatic ability. Young's modulus changes following treatment with the anti-cancer drugs paclitaxel, cisplatin and dacarbazine were also measured, offering a novel perspective through the use of continuous scan mode SICM. We found that Young's modulus was inversely correlated to metastatic ability in melanoma cell lines from radial growth, vertical growth and metastatic phases. However, Young's modulus was found to be highly variable between cells and cell lines. For example, the highly metastatic cell line A375M was found to have a significantly higher Young's modulus, and this was attributed to a higher level of F-actin. Furthermore, our data following nanomechanical changes after 24 hour anti-cancer drug treatment showed that paclitaxel and cisplatin treatment significantly increased Young's modulus, attributed to an increase in microtubules. Treatment with dacarbazine saw a decrease in Young's modulus with a significantly lower F-actin corrected total cell fluorescence. Our data offer a new perspective on nanomechanical changes following drug treatment, which may be an overlooked effect. This work also highlights variations in cell nanomechanical properties between previous studies, cancer cell lines and cancer types and questions the usefulness of using nanomechanics as a diagnostic or prognostic tool.