Volume Regulation and Nonosmotic Volume of Individual Human Platelets Quantified by High-Speed Scanning Ion Conductance Microscopy.

Background:  Platelets are anucleate cells that play an important role in wound closure following vessel injury. Maintaining a constant platelet volume is critical for platelet function. For example, water-induced swelling can promote procoagulant activity and initiate thrombosis.

The structure and mechanics of the cell cortex depend on the location and adhesion state.

Cells exist in different phenotypes and can transition between them. A phenotype may be characterized by many different aspects. Here, we focus on the example of whether the cell is adhered or suspended and choose particular parameters related to the structure and mechanics of the actin cortex.

Measuring the Shape, Stiffness, and Interface Tension of Droplets with the Scanning Ion Conductance Microscope.

Imaging and probing liquid-liquid interfaces at the micro- and nanoscale are of high relevance, for example, in materials science, surface chemistry, and microfluidics. However, existing imaging techniques are limited in resolution, average over large sample areas, or interact with the sample. Here, we present a method to quantify the shape, stiffness, and interface tension of liquid droplets with the scanning ion conductance microscope (SICM), providing submicrometer resolution and the ability to perform noncontact mechanical measurements.

Homophilic Interaction Between Transmembrane-JAM-A and Soluble JAM-A Regulates Thrombo-Inflammation: Implications for Coronary Artery Disease.

Genetic predisposition through single-nucleotide variation (SNV) influences circulatory soluble junctional adhesion molecule-A (sJAM-A) levels in coronary artery disease (CAD) patients. Homozygous carriers of the minor alleles (SNVs rs2774276, rs790056) show enhanced levels of thrombo-inflammatory sJAM-A. Both SNVs and sJAM-A are associated with worse prognosis for recurrent myocardial infarction in CAD patients.

Mechanics of migrating platelets investigated with scanning ion conductance microscopy.

Platelets are small blood cells involved in hemostasis, wound healing, and immune response. After adhesion and spreading, platelets can migrate at sites of injury inducing an early immune response to inflammation or infection. Platelet migration requires fibrinogen-integrin binding and fibrinogen depletion from the substrate inducing a self-generated ligand gradient guiding the direction of migration.

Spatial correlation of cell stiffness and traction forces in cancer cells measured with combined SICM and TFM.

The mechanical properties of cancer cells at the single-cell and the subcellular level might be the key for answering long-standing questions in the diagnosis and treatment of cancer. However, the subcellular distribution of two main mechanical properties, cell stiffness and traction forces, has been investigated only rarely and qualitatively yet. Here, we present the first direct combination of scanning ion conductance microscopy (SICM) and traction force microscopy (TFM), which we used to identify a correlation between the local stiffness and the local traction force density in living cells.

Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation.

Cortical cell stiffness is independent of substrate mechanics.

Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell.

Mapping the creep compliance of living cells with scanning ion conductance microscopy reveals a subcellular correlation between stiffness and fluidity.

Living cells exhibit complex material properties, which play a crucial role in many aspects of cell function in health and disease, including migration, proliferation, differentiation, and apoptosis. Various techniques exist to probe the viscoelastic material properties of living cells and a frequent observation is a cell-to-cell correlation between average stiffness and fluidity in populations of cells. However, the origin of this correlation is still under discussion.

Combined atomic force microscopy (AFM) and traction force microscopy (TFM) reveals a correlation between viscoelastic material properties and contractile prestress of living cells.

Living cells exhibit a complex mechanical behavior, whose underlying mechanisms are still largely unknown. Emerging from the molecular structure and dynamics of the cytoskeleton, the mechanical behavior comprises "passive" viscoelastic material properties and "active" contractile prestress. To directly investigate the connection between these quantities at the single-cell level, we here present the combination of atomic force microscopy (AFM) with traction force microscopy (TFM).

Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

Background: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant.

An Accurate Model for the Ion Current-Distance Behavior in Scanning Ion Conductance Microscopy Allows for Calibration of Pipet Tip Geometry and Tip-Sample Distance.

The scanning ion conductance microscope (SICM) is an emerging tool for noncontact topography imaging and multiphysical investigation of soft samples in aqueous environments such as living cells. Despite the increasing popularity of SICM, several aspects of the imaging process are still unknown; for example, there is still no accurate description of the behavior of the ion current for a varying tip-sample distance. To predict this ion current-distance behavior, we provide a new numerical model based on finite element modeling.

Thrombin-induced cytoskeleton dynamics in spread human platelets observed with fast scanning ion conductance microscopy.

Platelets are small anucleate blood cells involved in haemostasis. Platelet activation, caused by agonists such as thrombin or by contact with the extracellular matrix, leads to platelet adhesion, aggregation, and coagulation. Activated platelets undergo shape changes, adhere, and spread at the site of injury to form a blood clot.

Regulation of oxidized platelet lipidome: implications for coronary artery disease.

Aims: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets.

Methods And Results: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.

Viscoelastic properties of normal and cancerous human breast cells are affected differently by contact to adjacent cells.

Unlabelled: Malignant transformation drastically alters the mechanical properties of the cell and its response to the surrounding cellular environment. We studied the influence of the physical contact between adjacent cells in an epithelial monolayer on the viscoelastic behavior of normal MCF10A, non-invasive cancerous MCF7, and invasive cancerous MDA-MB-231 human breast cells. Using an atomic force microscopy (AFM) imaging technique termed force clamp force mapping (FCFM) to record images of the viscoelastic material properties, we found that normal MCF10A cells are stiffer and have a lower fluidity at confluent than at sparse density.

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets.

Platelet-derived HMGB1 is a critical mediator of thrombosis.

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis.

Lateral Resolution and Image Formation in Scanning Ion Conductance Microscopy.

The scanning ion conductance microscope (SICM) is a powerful tool for imaging the topography of soft samples in an aqueous environment. Despite the rising popularity of the SICM, the image formation process and the fundamental limit of the lateral resolution are still a matter of debate. Using microfabricated samples, we investigated the imaging of small cylindrical particles, elongated objects, and topography steps and present the first direct comparison of numerical and experimental data.

Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging.

Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant.

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted.

Imaging the elastic modulus of human platelets during thrombin-induced activation using scanning ion conductance microscopy.

Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated.

High-frequency ultrasound-guided disruption of glycoprotein VI-targeted microbubbles targets atheroprogressison in mice.

Targeted contrast-enhanced ultrasound (CEU) using microbubble agents is a promising non-invasive imaging technique to evaluate atherosclerotic lesions. In this study, we decipher the diagnostic and therapeutic potential of targeted-CEU with soluble glycoprotein (GP)-VI in vivo. Microbubbles were conjugated with the recombinant fusion protein GPVI-Fc (MBGPVI) that binds with high affinity to atherosclerotic lesions.

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.

High-speed force mapping on living cells with a small cantilever atomic force microscope.

The imaging speed of the wide-spread force mapping mode for quantitative mechanical measurements on soft samples in liquid with the atomic force microscope (AFM) is limited by the bandwidth of the z-scanner and viscous drag forces on the cantilever. Here, we applied high-speed, large scan-range atomic force microscopy and small cantilevers to increase the speed of force mapping by ≈10-100 times. This allowed resolving dynamic processes on living mouse embryonic fibroblasts.

Bacterial interactions with proteins and cells relevant to the development of life-threatening endocarditis studied by use of a quartz-crystal microbalance.

Implant-related infections are a major challenge in clinical routine because of severe complications, for example infective endocarditis (IE). The purpose of this study was to investigate the real-time interaction of S. gordonii with proteins and cells important in the development of IE, in a flow system, by use of a quartz-crystal microbalance (QCM).