Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

Implantation of defined activities of phosphorus 32 with reduced target damage.

Materials doped with the unstable isotope phosphorus 32 are promising candidates for use in brachytherapeutic applications. One way to dope a material with 32P is by ion implantation. However, the bombardment of the target with ions other than 32P due to impurities of the ion beam leads to unnecessary damages of the target, which might reduce its potential for medical applications. Furthermore, implanting a pre-selected activity of an unstable isotope into a target requires the repeated determination of the target's activity, which requires removing the target from the implantation chamber. This prolongs the total implantation time and requires handling the radioactive target multiple times, which in turn increases the risk of accidental exposure. We have incorporated an online-detector system into the implantation chamber of a 60 kV ion implanter that allowed us to determine the activity of the target without removing the target from the implantation chamber. We then used this system to investigate the implantation of ions with m = 38 u-instead of ions with m = 32 u-to reduce the fraction of other ions than 32P implanted into the target to reduce the induced damages.

Correlative Stimulated Emission Depletion and Scanning Ion Conductance Microscopy.

Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and nonphysiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, scanning ion conductance microscopy (SICM) has been shown to be the technique that minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is stimulated emission depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and SICM. We show that both methods allow recording a living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a subdiffraction fluorescence method and might form the basis for the development of a combined instrument that would allow the simultaneous recording of subdiffraction fluorescence and topography information.

A low-cost, large field-of-view scanning ion conductance microscope for studying nanoparticle-cell membrane interactions.

Nanoparticles have the potential to become versatile tools in the medical and life sciences. One potential application is delivering drugs or other compounds to the cell cytoplasm, which requires the nanoparticles to bind to or cross the cell membrane. However, there are only a few tools available which allow studying the interaction of nanoparticles and the cell membrane of living cells in a physiological environment. Currently, the tool which least biases living cells is Scanning Ion Conductance Microscopy (SICM). Specialized SICMs allow imaging at high resolution, however, they are cost intensive, particularly when providing a large field-of-view. In contrast, less cost intensive SICMs which provide a large field-of-view do not allow imaging at high resolutions. We have developed a SICM setup consisting of a compact three-axis piezo system and an additional fast shear-force piezo actor. This combination allows imaging fields-of-view of up to 80 µm × 80 µm, recording sections of living cells with a temporal resolution in the range of minutes as well as imaging with a spatial resolution of below 70 nm. Using our SICM we found that the cell membrane of HeLa cells treated with carboxylated latex nanoparticles was significantly more convoluted compared to control cells. The SICM setup we introduce here combines high resolution imaging with a large field-of-view at low costs. Our setup only requires a mounting adapter to extend existing inverted light microscopes, thus it could be a valuable and cost effective tool for researchers in all fields of the medical and life sciences performing investigations at the nanometer scale.

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.

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.

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.

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.

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.

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

BACKGROUND: Cell 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. RESULTS: We 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. CONCLUSIONS: We 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.

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.