Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification.

Reflection interference contrast microscopy (RICM) allows the visualization of the cell's adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell's spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function.

This study describes a novel assay to visualize the macromolecular permeability of epithelial and endothelial cell layers with subcellular lateral resolution. Defects within the cell layer and details about the permeation route of the migrating solute are revealed. The assay is based on silicon chips with densely packed, highly ordered, dead-ended pores of µm-diameters on one side. The cells under study are grown on the porous side of the chip such that the pores in the growth surface serve as an array of femtolitre-sized cuvettes in which the permeating probe accumulates at the site of permeation. The pattern of pore filling reveals the permeability characteristics of the cell layer with a lateral resolution in the µm range. Coating of the chip surface with a thin layer of gold allows for impedance analysis of the adherent cells in order to measure their tightness for inorganic ions at the same time. The new assay provides an unprecedented look on epithelial and endothelial barrier function.

Simplified approach for quantitative digital holographic phase contrast imaging of living cells.

Many interferometry-based quantitative phase contrast imaging techniques require a separately generated coherent reference wave. This results in a low phase stability and the demand for a precise adjustment of the intensity ratio between object and reference wave. To overcome these problems, the performance of a Michelson interferometer approach for digital holographic microscopy was analyzed that avoids a separately generated reference wave by superposition of different image areas. It is shown that this simplified arrangement yields improved phase stability. Furthermore, results from time-lapse investigations on living pancreas tumor cells demonstrate the capability of the method for reliable quantitative phase contrast imaging.

Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays.

BACKGROUND: Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. RESULTS: Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. CONCLUSION: In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints.

Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index.

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.

Elasticity mapping of pore-suspending native cell membranes.

The mechanics of cellular membranes are governed by a non-equilibrium composite framework consisting of the semiflexible filamentous cytoskeleton and extracellular matrix proteins linked to the lipid bilayer. While elasticity information of plasma membranes has mainly been obtained from whole cell analysis, techniques that allow addressing local mechanical properties of cell membranes are desirable to learn how their lipid and protein composition is reflected in the elastic behavior on local length scales. Introduced here is an approach based on basolateral membranes of polar epithelial Madin-Darby canine kidney (MDCK) II cells, prepared on a highly ordered porous substrate that allows elastic mapping on a submicrometer-length scale. A strong correlation between the density of actin filaments and the measured membrane elasticity is found. Spatially resolved indentation experiments carried out with atomic force and fluorescence microscope permit relation of the supramolecular structure to the elasticity of cellular membranes. It is shown that the elastic response of the pore spanning cell membranes is governed by local bending modules rather than lateral tension.

Novel template-directed anodic phenol-coupling reaction.

Substituted phenols were anodically coupled to the corresponding 2,2'-biphenols via tetraphenoxy borate derivatives. This electrochemical method is particularly useful for methyl-substituted substrates, such as 2,4-dimethyl phenol. The selective ortho-coupling reaction can be easily performed on a multikilogram scale.