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 We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances.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.

Live-seq enables temporal transcriptomic recording of single cells.

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Dissecting cell membrane tension dynamics and its effect on Piezo1-mediated cellular mechanosensitivity using force-controlled nanopipettes.

The dynamics of cellular membrane tension and its role in mechanosensing, which is the ability of cells to respond to physical stimuli, remain incompletely understood, mainly due to the lack of appropriate tools. Here, we report a force-controlled nanopipette-based method that combines fluidic force microscopy with fluorescence imaging for precise manipulation of the cellular membrane tension while monitoring the impact on single-cell mechanosensitivity. The force-controlled nanopipette enables control of the indentation force imposed on the cell cortex as well as of the aspiration pressure applied to the plasma membrane. We show that this setup can be used to concurrently monitor the activation of Piezo1 mechanosensitive ion channels via calcium imaging. Moreover, the spatiotemporal behavior of the tension propagation is assessed with the fluorescent membrane tension probe Flipper-TR, and further dissected using molecular dynamics modeling. Finally, we demonstrate that aspiration and indentation act independently on the cellular mechanobiological machinery, that indentation induces a local pre-tension in the membrane, and that membrane tension stays confined by links to the cytoskeleton.

Mitochondria transplantation between living cells.

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. The technology combines atomic force microscopy, optical microscopy, and nanofluidics to achieve force and volume control with real-time inspection. We developed dedicated probes that allow minimally invasive entry into cells and optimized fluid flow to extract specific organelles. When extracting single or a defined number of mitochondria, their morphology transforms into a pearls-on-a-string phenotype due to locally applied fluidic forces. We show that the induced transition is calcium independent and results in isolated, intact mitochondria. Upon cell-to-cell transplantation, the transferred mitochondria fuse to the host cells mitochondrial network. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed monitoring of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 minutes after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, donor mitochondrial DNA (mtDNA) was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for therapy, mechanobiology, and synthetic biology.

Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

4D Force Detection of Cell Adhesion and Contractility.

Mechanical signals establish two-way communication between mammalian cells and their environment. Cells contacting a surface exert forces via contractility and transmit them at the areas of focal adhesions. External stimuli, such as compressive and pulling forces, typically affect the adhesion-free cell surface. Here, we demonstrate the collaborative employment of Fluidic Force Microscopy and confocal Traction Force Microscopy supported by the Cellogram solver to enable a powerful integrated force probing approach, where controlled vertical forces are applied to the free surface of individual cells, while the concomitant deformations are used to map their transmission to the substrate. Force transmission across human cells is measured with unprecedented temporal and spatial resolution, enabling the investigation of the cellular mechanisms involved in the adaptation, or maladaptation, to external mechanical stimuli. Altogether, the system enables facile and precise force interrogation of individual cells, with the capacity to perform population-based analysis.

Electrochemical 3D printing of Ni-Mn and Ni-Co alloy with FluidFM.

Additive manufacturing can realize almost any designed geometry, enabling the fabrication of innovative products for advanced applications. Local electrochemical plating is a powerful approach for additive manufacturing of metal microstructures; however, previously reported data have been mostly obtained with copper, and only a few cases have been reported with other elements. In this study, we assessed the ability of fluidic force microscopy to produce Ni-Mn and Ni-Co alloy structures. Once the optimal deposition potential window was determined, pillars with relatively smooth surfaces were obtained. The printing process was characterized by printing rates in the range of 50-60 nm s-1. Cross-sections exposed by focused ion beam showed highly dense microstructures, while the corresponding face scan with energy-dispersive x-ray spectroscopy spectra revealed a uniform distribution of alloy components.

Bringing Electrochemical Three-Dimensional Printing to the Nanoscale.

Nanoscale 3D printing is attracting attention as an alternative manufacturing technique for a variety of applications from electronics and nanooptics to sensing, nanorobotics, and energy storage. The constantly shrinking critical dimension in state-of-the-art technologies requires fabrication of complex conductive structures with nanometer resolution. Electrochemical techniques are capable of producing impurity-free metallic conductors with superb electrical and mechanical properties, however, true nanoscale resolution (<100 nm) remained unattainable. Here, we set new a benchmark in electrochemical 3D printing. By employing nozzles with dimensions as small as 1 nm, we demonstrate layer-by-layer manufacturing of 25 nm diameter voxels. Full control of the printing process allows adjustment of the feature size on-the-fly, printing tilted, and overhanging structures. On the basis of experimental evidence, we estimate the limits of electrochemical 3D printing and discuss the origins of this new resolution frontier.

Mechanical Fingerprint of Senescence in Endothelial Cells.

Endothelial senescence entails alterations of the healthy cell phenotype, which accumulate over time and contribute to cardiovascular disease. Mechanical aspects regulating cell adhesion, force generation, and the response to flow contribute to the senescence-associated drift; however, they remain largely unexplored. Here, we exploit force microscopy to resolve variations of the cell anchoring to the substrate and the tractions generated upon aging in the nanonewton (nN) range. Senescent endothelial cells display a multifold increase in the levels of basal adhesion and force generation supported by mature and strong focal adhesions. The enhanced mechanical interaction with the substrate yields static endothelial monolayers that polarize in response to flow but fail the process of coordinated cell shape remodeling and reorientation. The emerging picture indicates that senescence reinforces the local cell interaction with the substrate and may therefore prevent endothelial denudation; however, it compromises the ability to functionally adapt to the local hemodynamic conditions.

Metals by Micro-Scale Additive Manufacturing: Comparison of Microstructure and Mechanical Properties.

Many emerging applications in microscale engineering rely on the fabrication of 3D architectures in inorganic materials. Small-scale additive manufacturing (AM) aspires to provide flexible and facile access to these geometries. Yet, the synthesis of device-grade inorganic materials is still a key challenge toward the implementation of AM in microfabrication. Here, a comprehensive overview of the microstructural and mechanical properties of metals fabricated by most state-of-the-art AM methods that offer a spatial resolution ≤10 µm is presented. Standardized sets of samples are studied by cross-sectional electron microscopy, nanoindentation, and microcompression. It is shown that current microscale AM techniques synthesize metals with a wide range of microstructures and elastic and plastic properties, including materials of dense and crystalline microstructure with excellent mechanical properties that compare well to those of thin-film nanocrystalline materials. The large variation in materials' performance can be related to the individual microstructure, which in turn is coupled to the various physico-chemical principles exploited by the different printing methods. The study provides practical guidelines for users of small-scale additive methods and establishes a baseline for the future optimization of the properties of printed metallic objects-a significant step toward the potential establishment of AM techniques in microfabrication.

Elasticity spectra as a tool to investigate actin cortex mechanics.

BACKGROUND: The mechanical properties of single living cells have proven to be a powerful marker of the cell physiological state. The use of nanoindentation-based single cell force spectroscopy provided a wealth of information on the elasticity of cells, which is still largely to be exploited. The simplest model to describe cell mechanics is to treat them as a homogeneous elastic material and describe it in terms of the Young's modulus. Beside its simplicity, this approach proved to be extremely informative, allowing to assess the potential of this physical indicator towards high throughput phenotyping in diagnostic and prognostic applications. RESULTS: Here we propose an extension of this analysis to explicitly account for the properties of the actin cortex. We present a method, the Elasticity Spectra, to calculate the apparent stiffness of the cell as a function of the indentation depth and we suggest a simple phenomenological approach to measure the thickness and stiffness of the actin cortex, in addition to the standard Young's modulus. CONCLUSIONS: The Elasticity Spectra approach is tested and validated on a set of cells treated with cytoskeleton-affecting drugs, showing the potential to extend the current representation of cell mechanics, without introducing a detailed and complex description of the intracellular structure.

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.

Local Chemical Stimulation of Neurons with the Fluidic Force Microscope (FluidFM).

Physiological communication between neurons is dependent on the exchange of neurotransmitters at the synapses. Although this chemical signal transmission targets specific receptors and allows for subtle adaptation of the action potential, in vitro neuroscience typically relies on electrical currents and potentials to stimulate neurons. The electric stimulus is unspecific and the confinement of the stimuli within the media is technically difficult to control and introduces large artifacts in electric recordings of the activity. Here, we present a local chemical stimulation platform that resembles in vivo physiological conditions and can be used to target specific receptors of synapses. Neurotransmitters were dispensed using the force-controlled fluidic force microscope (FluidFM) nanopipette, which provides exact positioning and precise liquid delivery. We show that controlled release of the excitatory neurotransmitter glutamate induces spiking activity in primary rat hippocampal neurons, as measured by concurrent electrical and optical recordings using a microelectrode array and a calcium-sensitive dye, respectively. Furthermore, we characterized the glutamate dose response of neurons by applying stimulation pulses of glutamate with concentrations from 0 to 0.5 mm. This new stimulation approach, which combines FluidFM for gentle and precise positioning with a microelectrode array read-out, makes it possible to modulate the activity of individual neurons chemically and simultaneously record their induced activity across the entire neuronal network. The presented platform not only offers a more physiological alternative compared with electrical stimulation, but also provides the possibility to study the effects of the local application of neuromodulators and other drugs.

Pattern Formation in Binary Colloidal Assemblies: Hidden Symmetries in a Kaleidoscope of Structures.

In this study, we present a detailed investigation of the morphology of binary colloidal structures formed by self-assembly at air/water interface of particles of two different sizes, with a size ratio such that the larger particles do not retain a hexagonal arrangement in the binary assembly. While the structure and symmetry of binary mixtures in which such hexagonal order is preserved has been thoroughly scrutinized, binary colloids in the regime of nonpreservation of the hexagonal order have not been examined with the same level of detail due also to the difficulty in finding analysis tools suitable to recognize hidden symmetries in seemingly amorphous and disordered arrangements. For this purpose, we resorted to a combination of different analysis tools based on computational geometry and computational topology to get a comprehensive picture of the morphology of the assemblies. By carrying out an extensive investigation of binary assemblies in this regime with variable concentration of smaller particles with respect to larger particles, we identify the main patterns that coexist in the apparently disordered assemblies and detect transitions in the symmetries upon increase in the number of small particles. As the concentration of small particles increases, large particle arrangements become more dilute and a transition from hexagonal to rhombic and square symmetries occurs, accompanied also by an increase in clusters of small particles; the relative weight of each specific symmetry can be controlled by varying the composition of the assemblies. The demonstration of the possibility to control the morphology of apparently disordered binary colloidal assemblies by varying experimental conditions and the definition of a route for the investigation of disordered assemblies are important for future studies of complex colloidal patterns to understand self-assembly mechanisms and to tailor the physical properties of colloidal assemblies.

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.

Self-Assembly of Single-Sized and Binary Colloidal Particles at Air/Water Interface by Surface Confinement and Water Discharge.

We present an innovative apparatus allowing self-assembly at air/water interface in a smooth and reproducible way. The combination of water discharge and surface confinement of the area over which self-assembly takes place allows transfer of the assembled monolayer without any risk of damage to the colloidal crystal. As we demonstrate, the designed approach offers remarkable advantages in terms of cost and robustness compared to state-of-the art methods and is suitable for the fabrication of highly ordered monolayers even for more challenging assembly experiments such as transfer on rough substrates or assembly of binary colloids. Hence, our apparatus represents a significant headway toward high scale production of large area colloidal crystals. For the binary colloid assembly experiments, we also report the first experimental demonstration of a morphology based on the alternation of three and four small particles in the interstices between large particles.

Serial weighting of micro-objects with resonant microchanneled cantilevers.

Atomic force microscopy (AFM) cantilevers have proven to be very effective mass sensors. The attachment of a small mass to a vibrating cantilever produces a resonance frequency shift that can be monitored, providing the ability to measure mass changes down to a few molecules resolution. Nevertheless, the lack of a practical method to handle the catch and release process required for dynamic weighting of microobjects strongly hindered the application of the technology beyond proof of concept measurements. Here, a method is proposed in which FluidFM hollow cantilevers are exploited to overcome the standard limitations of AFM-based mass sensors, providing high throughput single object weighting with picogram accuracy. The extension of the dynamic models of AFM cantilevers to hollow cantilevers was discussed and the effectiveness of mass weighting in air was validated on test samples.

Adsorption of single 1,8-octanedithiol molecules on Cu(100).

Single 1,8-octanedithiol (ODT) molecules adsorbed onto the Cu(100) surface have been characterized by using scanning tunneling microscopy (STM) and studied by semi-empirical calculations. STM images have revealed two types of chiral molecules on the surface upon adsorption and both types of molecules showed two bright spots at the extremities of a small rod due to the enhanced electronic density contrast of the chemisorbed sulfur atoms. In sub-monolayer regime deposition, ODT molecules exhibit preferential adsorption directions and the relaxation mechanism is driven by the chemisorption of the two sulfur atoms in a hollow site of the surface. By means of calculations several conformations of the molecule according to the energetically favorable alkane body stretching constraint have been studied. The comparison between relaxed conformations and between calculated and experimental STM images, followed by an analysis of different orientations, has allowed determining unambiguously the most favorable position of the ODT molecule on Cu(100).

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

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.