Salvatore Girardo

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.

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.

Three-dimensional correlative single-cell imaging utilizing fluorescence and refractive index tomography

Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.

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.

Measuring Microtubule Supertwist and Defects by 3D-Force-Clamp Tracking of Single Kinesin-1 Motors

Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it is hypothesized that microtubule supertwist and defects can severely influence the function of motors and other microtubule-associated proteins, the presented method allows for the first time to fully map the microtubule lattice in situ. This mapping allows the correlation of motor-filament interactions with the microtubule fine-structure. With the additional ability to apply loads, we expect our 3D-optical-tweezers force clamp to become a valuable tool for obtaining a wide range of information from other biological systems, inaccessible by two-dimensional and/or ensemble measurements.

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.

Directionally biased sidestepping of Kip3/kinesin-8 is regulated by ATP waiting time and motor–microtubule interaction strength

Significance Microtubules consist of parallel protofilament lanes for motor-based intracellular transport. While most motors translocate along individual protofilaments, members of the kinesin-8 family have been reported to sidestep. However, the mechanism of sidestepping is currently not understood. Here, we track the 3D motion of single kinesin-8 motors on freely suspended microtubules. We find that individual motors sidestep with a bias to the left, the probability of which is increased with the time taken per forward step. We relate this behavior to a bifurcation in the step cycle of kinesin-8. Experiments on a kinesin-1 with an elongated neck linker suggest that sidestepping is intrinsic to all processive kinesins possessing long neck linkers, potentially helping them to circumnavigate obstacles on the microtubule. Kinesin-8 motors, which move in a highly processive manner toward microtubule plus ends where they act as depolymerases, are essential regulators of microtubule dynamics in cells. To understand their navigation strategy on the microtubule lattice, we studied the 3D motion of single yeast kinesin-8 motors, Kip3, on freely suspended microtubules in vitro. We observed short-pitch, left-handed helical trajectories indicating that kinesin-8 motors frequently switch protofilaments in a directionally biased manner. Intriguingly, sidestepping was not directly coupled to forward stepping but rather depended on the average dwell time per forward step under limiting ATP concentrations. Based on our experimental findings and numerical simulations we propose that effective sidestepping toward the left is regulated by a bifurcation in the Kip3 step cycle, involving a transition from a two-head–bound to a one-head–bound conformation in the ATP-waiting state. Results from a kinesin-1 mutant with extended neck linker hint toward a generic sidestepping mechanism for processive kinesins, facilitating the circumvention of intracellular obstacles on the microtubule surface.

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.