Patrick Happel

Monitoring cell movements and volume changes with pulse‐mode scanning ion conductance microscopy

Here we describe the use of pulse‐mode scanning ion conductance microscopy (SICM) to observe volume changes and cell membrane movements during the locomotion of cultured cells in the range of minutes to several hours. The microscope is based on the pulse‐mode SICM previously developed for stable imaging of single cells in culture. Our instrument uses current pulses to control the distance between cell surface and electrode tip as well as a back‐step mode to prevent contact of tip and membrane during lateral movements of the probe. We performed repeated scans of cell surfaces using feedback‐controlled piezoactors to position the electrode. Using patch‐clamp‐type electrode tips the height of cells could reproducibly be measured with a standard deviation of 50 nm. To quantify and separate changes in cell position and volume occurring between consecutive scans, a program was written to subtract images and calculate volume changes. Examples of repeated scans show that membrane movements in the range of 30 min to a few hours can be quantitatively monitored with a lateral resolution of 500 nm using difference images and that faster movements in the range of minutes can be recorded at defined cell sections using the line scan mode. Difference images indicate that volume changes can affect cell surfaces inhomogeneously, emphazising the role of the cytoskeleton in the stabilization of cell shape.

Backstep scanning ion conductance microscopy as a tool for long term investigation of single living cells

Scanning ion conductance microscopy (SICM) is a suitable tool for imaging surfaces of living cells in a contact-free manner. We have shown previously that SICM in backstep mode allows one to trace the outlines of entire cell somata and to detect changes in cellular shape and volume. Here we report that SICM can be employed to quantitatively observe cellular structures such as cell processes of living cells as well as cell somata of motile cells in the range of hours.

Scanning ion conductance microscopy for studying biological samples.

Scanning ion conductance microscopy (SICM) is a scanning probe technique that utilizes the increase in access resistance that occurs if an electrolyte filled glass micro-pipette is approached towards a poorly conducting surface. Since an increase in resistance can be monitored before the physical contact between scanning probe tip and sample, this technique is particularly useful to investigate the topography of delicate samples such as living cells. SICM has shown its potential in various applications such as high resolution and long-time imaging of living cells or the determination of local changes in cellular volume. Furthermore, SICM has been combined with various techniques such as fluorescence microscopy or patch clamping to reveal localized information about proteins or protein functions. This review details the various advantages and pitfalls of SICM and provides an overview of the recent developments and applications of SICM in biological imaging. Furthermore, we show that in principle, a combination of SICM and ion selective micro-electrodes enables one to monitor the local ion activity surrounding a living cell.

Maskless and targeted creation of arrays of colour centres in diamond using focused ion beam technology

The creation of nitrogen-vacancy (NV) centres in diamond is nowadays well controlled using nitrogen implantation and annealing. Although the high-resolution placement of NV centres has been demonstrated using either collimation through pierced tips of an atomic force microscope (AFM) or masks with apertures made by electron beam lithography, a targeted implantation into pre-defined structures in diamond may not be achieved using these techniques. We show that a beam of nitrogen ions can be focused to approximately 100 nm using focused ion beam (FIB) technology. The nitrogen ion beam is produced using an electron cyclotron resonance (ECR) plasma source. Combined with a scanning electron microscope, the nitrogen-FIB offers new possibilities for the targeted creation of single defects in diamond. This maskless technology is suitable for example for the creation of optical centres in the cavities of photonic crystals or in diamond tips for scanning magnetometry.

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.

Fast rearrangement of the neuronal growth cone’s actin cytoskeleton following VEGF stimulation

The neuronal growth cone plays a crucial role in the development of the nervous system. This highly motile structure leads the axon to its final destination by translating guidance cues into cytoskeletal rearrangements. Recently, vascular endothelial growth factor (VEGF), which is essential for angiogenesis and vascular sprouting, has been found to exert a trophic activity also on neurons, leading to an increased axonal outgrowth, similar to the well-known nerve growth factor (NGF). The neurotrophic properties of VEGF are likely to be promoted via the VEGF receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1). In the long term, VEGF attracts and influences the growth cone velocity and leads to growth cone enlargement. The present study focuses on immediate VEGF effects using RFP-actin and GFP-NF-M microinjected chicken dorsal root ganglia for live cell imaging of the neuronal growth cone. We analyzed actin and neurofilament dynamics following VEGF and NGF treatment and compared the effects. Furthermore, key signaling pathways of VEGF were investigated by specific blocking of VEGFR-2 or NRP-1. With the aid of confocal laser scanning microscopy and stimulated emission depletion microscopy, we show for the first time that VEGF has a quick effect on the actin-cytoskeleton, since actin rearrangements were identifiable within a few minutes, leading to a dramatically increased motion. Moreover, these effects were strongly enhanced by adding both VEGF and NGF. Most notably, the effects were inhibited by blocking VEGFR-2, therefore we propose that the immediate effects of VEGF on the actin-cytoskeleton are mediated through VEGFR-2.

A boundary delimitation algorithm to approximate cell soma volumes of bipolar cells from topographical data obtained by scanning probe microscopy

BackgroundCell volume determination plays a pivotal role in the investigation of the biophysical mechanisms underlying various cellular processes. Whereas light microscopy in principle enables one to obtain three dimensional data, the reconstruction of cell volume from z-stacks is a time consuming procedure. Thus, three dimensional topographic representations of cells are easier to obtain by scanning probe microscopical measurements.ResultsWe present a method of separating the cell soma volume of bipolar cells in adherent cell cultures from the contributions of the cell processes from data obtained by scanning ion conductance microscopy. Soma volume changes between successive scans obtained from the same cell can then be computed even if the cell is changing its position within the observed area. We demonstrate that the estimation of the cell volume on the basis of the width and the length of a cell may lead to erroneous determination of cell volume changes.ConclusionsWe provide a new algorithm to repeatedly determine single cell soma volume and thus to quantify cell volume changes during cell movements occuring over a time range of hours.

Migrating Oligodendrocyte Progenitor Cells Swell Prior to Soma Dislocation

The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma.

Long-term, long-distance recording of a living migrating neuron by scanning ion conductance microscopy

Bias-free, three-dimensional imaging of entire living cellular specimen is required for investigating shape and volume changes that occur during cellular growth or migration. Here we present fifty consecutive recordings of a living cultured neuron from a mouse dorsal root ganglion obtained by Scanning ion conductance microscopy (SICM). We observed a saltatory migration of the neuron with a mean velocity of approximately 20 μm/h. These results demonstrate the non-invasiveness of SICM, which makes it unique among the scanning probe microscopes. In contrast to SICM, most scanning probe techniques require a usually denaturating preparation of the cells, or they exert a non-negligible force on the cellular membrane, impeding passive observation. Moreover, the present series of recordings demonstrates the potential use of SICM for the detailed investigation of cellular migration and membrane surface dynamics even of such delicate samples as living neurons.

Fast rearrangement of the neuronal growth cone's actincytoskeleton following VEGF stimulation.

Development of nervous system involves neuronal migration and axonal navigation. Growth cone which is a highly motile structure at the distal tip of growing axon leads to its final destination. Various guidance cues plays crucial role in cytoskeletal rearrangement by forming finger like structure filopodia and flat veil like lamellipodia. Out of these cues vascular endothelial growth factor (VEGF) is the putative factor for angiogenesis or vascular sprouting, also has trophic activity similar to the nerve growth factor (NGF) on neuronal cells. It also stimulates axonal growth and also enhances survival of dorsal root ganglion or cervical neurons. VEGF is known to inhibit hypoxic death of cortical neurons in-vitro2 and in cerebral ischemia. doi : 10.5214/ans.0972.7531.200307