Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Combined Ion Conductance and Atomic Force Microscope for Fast Simultaneous Topographical and Surface Charge Imaging.

We report here an advanced approach for simultaneous and independent submicroscale imaging of local surface charge and topography using microchanneled cantilevers, also known as FluidFM nanopipette probes. These hollow cantilevers with a 300 nm opening are employed for ion current measurements that provide access to the local properties of the electrical double layer using the phenomenon of ion current rectification, while also taking advantage of the force sensing capabilities for accurate probe vertical positioning and topography imaging. The independent nature of this atomic force microscope (AFM) feedback opens up a possibility to significantly increase the sensitivity for probing local surface charges in a wider range of salt concentrations, especially in electrolytes of low ionic strength (below 10 mM), where classical local ion conductance measurements with glass nanopipettes would suffer from inaccuracies and instabilities, but where the electrical double layer extends further into the liquid medium and has stronger effect on the measured ion currents for charge imaging. We demonstrate that the measurements with FluidFM do not compromise the positioning accuracy and enable accurate and simultaneous topographical and charge imaging in contact mode (similar to AFM) at high scanning rates, approaching thousands of pixels per second, therefore overtaking state-of-the-art techniques for charge mapping by at least 2 orders of magnitude (the probes reach translation rates of 120 µm s-1 equating to 2 ms per image pixel). We also reveal experimentally the physical limit of this high speed scanning, constrained by the rate of ion redistribution in surface-induced rectification required for double layer sensing and charge mapping.

Extending the limits of direct force measurements: colloidal probes from sub-micron particles.

Direct force measurements by atomic force microscopy (AFM) in combination with the colloidal probe technique are widely used to determine interaction forces in colloidal systems. However, a number of limitations are still preventing a more universal applicability of this technique. Currently, one of the most significant limitations is that only particles with diameters of several micrometers can be used as probe particles. Here, we present a novel approach, based on the combination of nanofluidics and AFM (also referred to as FluidFM-technique), that allows to overcome this size limit and extend the size of suitable probe particles below diameters of 500 nanometers. Moreover, by aspiration of colloidal particles with a hollow AFM-cantilever, the immobilization process is independent of the particle's surface chemistry. Furthermore, the probe particles can be exchanged in situ. The applicability of the FluidFM-technique is demonstrated with silica particles, which are also the types of particles most often used for the preparation of colloidal probes. By comparing 'classical' colloidal probes, i.e. probes from particles irreversibly attached with glue, and various particle sizes aspirated by the FluidFM-technique, we can quantitatively evaluate the instrumental limits. Evaluation of the force profiles demonstrate that even for 500 nm silica particles the diffuse layer properties can be evaluated quantitatively. Therefore, direct force measurements on the level of particle sizes used in industrial formulations will become available in the future.

Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy.

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in 13C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

Tunable Single-Cell Extraction for Molecular Analyses.

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level.

Template-Free 3D Microprinting of Metals Using a Force-Controlled Nanopipette for Layer-by-Layer Electrodeposition.

A novel 3D printing method for voxel-by-voxel metal printing is presented. Hollow atomic force microscopy (AFM) cantilevers are used to locally supply metal ions in an electrochemical cell, enabling a localized electroplating reaction. By exploiting the deflection feedback of these probes, electrochemical 3D metal printing is, for the first time, demonstrated in a layer-by-layer fashion, enabling the fabrication of arbitrary-shaped geometries.

Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers.

We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.