Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label‐free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM‐FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic β‐cells. Using SICM‐FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal

Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis

Nanoscale visualization of redox activity at lithium-ion battery cathodes

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 ∼100 €‰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. © 2014 Macmillan Publishers Limited. All rights reserved

Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization

Although action potentials propagate along axons in an all-­or-­none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog-­digital modulation is depolarization-­mediated inactivation of presynaptic Kv1-­family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures, and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of Episodic Ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1;; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-­associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies

Nanoscale live-cell imaging using hopping probe ion conductance microscopy,

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by the AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample, but true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been reported. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, which allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . Until now, SICM has been restricted to imaging relatively flat surfaces, as all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM, a nanopipette is mounted on a three-dimensional piezoelectric translation stage and automatic feedback control moves the pipette up or down to keep the pipette current constant (the set point) while the sample is scanned in x and y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features We illustrate the benefits of HPICM in In contrast to conventional raster scanning, HPICM has the additional advantage that the order of imaging pixels is not predetermined. Therefore, we divided the entire image into equal-sized square

Electrochemical Nanoprobes for Single-Cell Analysis

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5–200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells