Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies.

Intercalation and deintercalation of lithium ions at electrode surfaces are central to the operation of lithium-ion batteries. Yet, on the most important composite cathode surfaces, this is a rather complex process involving spatially heterogeneous reactions that have proved difficult to resolve with existing techniques. Here we report a scanning electrochemical cell microscope based approach to define a mobile electrochemical cell that is used to quantitatively visualize electrochemical phenomena at the battery cathode material LiFePO 4 , with resolution of B100 nm. The technique measures electrode topography and different electrochemical properties simultaneously, and the information can be combined with complementary microscopic techniques to reveal new perspectives on structure and activity. These electrodes exhibit highly spatially heterogeneous electrochemistry at the nanoscale, both within secondary particles and at individual primary nanoparticles, which is highly dependent on the local structure and composition.

The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.

Rationale Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. Objective To explore microdomain-targeted remodeling of ventricular L-type Ca2+ channels (LTCCs) in HF. Methods and Results Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore distribution of single LTCCs in different membrane microdomains of non-failing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to re-distribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability (Po) was dramatically increased (0.034±0.011 vs 0.154±0.027, P<0.001). High Po was linked to enhanced calcium-calmodulin kinase II (CaMKII)-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current which contributed to the development of early afterdepolarizations (EADs). A novel model of LTCC function in HF was developed; following its validation with experimental data, the model was used to ascertain how HF–induced T-tubule loss led to altered LTCC function and EADs. The HF myocyte model was then implemented in a 3D left ventricle model, demonstrating that such EADs can propagate and initiate reentrant arrhythmias. Conclusion Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electrical remodelling in HF and adds a new dimension to cardiovascular disease.

High‐resolution scanning electrochemical cell microscopy (SECCM) is used to image and quantitatively analyze the hydrogen evolution reaction (HER) catalytically active sites of 1H‐MoS2 nanosheets, MoS2, and WS2 heteronanosheets. Using a 20 nm radius nanopipette and hopping mode scanning, the resolution of SECCM was beyond the optical microscopy limit and visualized a small triangular MoS2 nanosheet with a side length of ca. 130 nm. The electrochemical cell provides local cyclic voltammograms with a nanoscale spatial resolution for visualizing HER active sites as electrochemical images. The HER activity difference of edge, terrace, and heterojunction of MoS2 and WS2 were revealed. The SECCM imaging directly visualized the relationship of HER activity and number of MoS2 nanosheet layers and unveiled the heterogeneous aging state of MoS2 nanosheets. SECCM can be used for improving local HER activities by producing sulfur vacancies using electrochemical reaction at the selected region.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.