Alexander Mietke

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.

Direct visualization of the subthalamic nucleus and its iron distribution using high-resolution susceptibility mapping

Histological studies have shown a relatively high iron concentration in the subthalamic nucleus (STN). T2‐ and T2*‐weighted sequences have previously been used to visualize the STN in vivo. The phase information of gradient‐echo images reflects the magnetic tissue properties more directly, e.g., iron is more paramagnetic than water. Unfortunately, phase images suffer from non‐local effects and orientation dependency. The goal of this study is to delineate the STN more precisely using susceptibility maps, calculated from phase images, which directly index magnetic tissue properties while removing the non‐local effects and orientation dependency. Use of 7T MRI enables high spatial resolution with good signal to noise ratio (SNR). Eight healthy subjects were scanned at 7T using a high‐resolution 3D gradient‐echo sequence. Susceptibility maps were calculated from phase data using a thresholding Fourier approach and a regularization approach using spatial priors. The susceptibility maps clearly distinguish the STN from the adjacent substantia nigra (SN). Their susceptibilities are quantitatively different (0.06 and 0.1 ppm for the STN and SN, respectively). These maps allowed the STN, SN, and the red nucleus to be manually segmented, thus providing 3D visualization of their boundaries. In sum, the STN can be more clearly distinguished from adjacent structures in susceptibility maps than in T2*‐weighted images or phase images. Hum Brain Mapp, 2012.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

High‐throughput cell mechanical phenotyping for label‐free titration assays of cytoskeletal modifications

The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label‐free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real‐time deformability cytometry (RT‐DC), a novel microfluidic tool to examine the effects of biochemically modified F‐actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half‐maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F‐actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT‐DC. Drug‐induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high‐throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label‐free dose‐response assays of cytoskeletal modifications.

Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing

Real-time deformability (RT-DC) is a method for high-throughput mechanical and morphological phenotyping of cells in suspension. While analysis rates exceeding 1000 cells per second allow for a label-free characterization of complex biological samples, e.g., whole blood, data evaluation has so far been limited to a few geometrical and material parameters such as cell size, deformation, and elastic Youngs modulus. But as a microscopy-based technology, RT-DC actually generates and yields multidimensional datasets that require automated and unbiased tools to obtain morphological and rheological cell information. Here, we present a statistical framework to shed light on this complex parameter space and to extract quantitative results under various experimental conditions. As model systems, we apply cell lines as well as primary cells and highlight more than 11 parameters that can be obtained from RT-DC data. These parameters are used to identify sub-populations in heterogeneous samples using Gaussian mixture models, to perform a dimensionality reduction using principal component analysis, and to quantify the statistical significance applying linear mixed models to datasets of multiple replicates.

Integrin-mediated attachment of the blastoderm to the vitelline envelope impacts gastrulation of insects

During gastrulation, physical forces reshape the simple embryonic tissue to form a complex body plan of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the tissue undergoing gastrulation movements is surrounded by a rigid protective shell4,5. While it is well recognized that gastrulation movements depend on forces generated by tissue-intrinsic contractility6,7, it is not known if interactions between the tissue and the protective shell provide additional forces that impact gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle Tribolium castaneum tightly adheres in a temporally coordinated manner to the vitelline envelope surrounding the embryo. This attachment generates an additional force that counteracts the tissue-intrinsic contractile forces to create asymmetric tissue movements. Furthermore, this localized attachment is mediated by a specific integrin, and its knock-down leads to a gastrulation phenotype consistent with complete loss of attachment. Moreover, analysis of another integrin in the fruit fly Drosophila melanogaster suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline. Together, our findings reveal a conserved mechanism whereby the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable counter-forces that shape gastrulation movements in insects.

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.