Christoph Herold

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Min protein patterns emerge from rapid rebinding and membrane interaction of MinE

In Escherichia coli, the pole-to-pole oscillation of the Min proteins directs septum formation to midcell, which is required for symmetric cell division. In vitro, protein waves emerge from the self-organization of MinD, a membrane-binding ATPase, and its activator MinE. For wave propagation, the proteins need to cycle through states of collective membrane binding and unbinding. Although MinD presumably undergoes cooperative membrane attachment, it is unclear how synchronous detachment is coordinated. We used confocal and single-molecule microscopy to elucidate the order of events during Min wave propagation. We propose that protein detachment at the rear of the wave, and the formation of the E-ring, are accomplished by two complementary processes: first, local accumulation of MinE due to rapid rebinding, leading to dynamic instability; and second, a structural change induced by membrane-interaction of MinE in an equimolar MinD–MinE (MinDE) complex, which supports the robustness of pattern formation.

Efficient Electroformation of Supergiant Unilamellar Vesicles Containing Cationic Lipids on ITO-Coated Electrodes

Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.

Long-range transport of giant vesicles along microtubule networks

We report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diameters of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, we were able to estimate the number of GFP-labeled motors involved in the transport of a single vesicle. We found that the vesicles were transported by the cooperative activity of typically 5-10 motor molecules. The presented assay is expected to open up further applications in the field of synthetic biology, aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnology, where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.

Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid–gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid–gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid–gel coexistence. We also discuss possible biological implications of our experimental findings.

Real-time fluorescence and deformability cytometry

The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.

Real-time fluorescence and deformability cytometry - flow cytometry goes mechanics

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument — opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.

Real-time deformability cytometry as a label-free indicator of cell function.

The mechanical properties of cells are known to be a label-free, inherent marker of biological function in health and disease. Wide-spread utilization has so far been impeded by the lack of a convenient measurement technique with sufficient throughput. To address this unmet need, we have recently introduced real-time deformability cytometry (RT-DC) for continuous mechanical single-cell classification of heterogeneous cell populations at rates of several hundred cells per second. Cells are driven through the constriction zone of a microfluidic chip leading to cell deformations due to hydrodynamic stresses only. Our custom-built image processing software performs image acquisition, image analysis and data storage on the fly. The ensuing deformations can be quantified and an analytical model enables the derivation of cell material properties. Performing RT-DC we highlight its potential to identify rare objects in heterogeneous suspensions and to track drug-induced changes in cells. In summary, RT-DC enables marker-free, quantitative phenotyping of heterogeneous cell populations with a throughput comparable to standard flow cytometry.

DNA Condensation at Freestanding Cationic Lipid Bilayers

We describe a previously unreported coil-globule transition of DNA electrostatically bound to a freestanding fluid cationic lipid membrane. The collapse of a DNA coil into a compact globule takes place after the DNA molecule attaches in an extended conformation to the membrane. DNA condensation is favored at a higher cationic lipid content, while at lower membrane charge densities coexistence of DNA random coils, partially collapsed conformations, and globules is observed.