Real-Time Deformability Cytometry
Deformability cytometry (DC) encompasses technologies that employ flow to induce cell deformation, enabling the characterization of their mechanical properties. Typically, cells are subjected to hydrodynamic stresses within microchannels, with high-speed cameras capturing images of the deformed cells. While traditional cell mechanics methods are limited to measuring a few hundred cells per hour, DC has dramatically increased throughput to thousands of cells per second, facilitating the clinical adoption of cell deformability assays (1).
Our group developed Real-time Deformability Cytometry (RT-DC), introduced by Otto et al. in 2015, a microfluidic technique designed to sample the mechanical properties of cells label-free in real-time and continuously at rates exceeding 3,000 events per second (2,3). Subsequent advancements by Rosendahl et al. included extensions for simultaneous fluorescence measurements (4), while Nawaz et al. added machine-learning-assisted sorting to enhance its capabilities (5).
In RT-DC, cells are flowing through a narrow microchannel slightly wider than their diameter, driven by syringe pumps. The cells get deformed by hydrodynamic stresses acting on the cell surface, and a snapshot of the cell is taken by a CMOS camera, imaged through an inverted microscope.
We quantify the deformation of a cell by computing how much the contour deviates from a perfect circle (see image below). From the size and deformation of the cells, we can compute the cell stiffness, quantified by the Young’s modulus (6–8).
RT-DC is capable of analyzing diluted whole blood samples, identifying major blood cell types without labeling or enrichment (9). It was already employed to detect changes in red blood cell deformability in conditions like spherocytosis or malaria infection, as well as alterations in red and white blood cell deformability in infectious diseases (9). Notably, our research revealed that neutrophils softened during acute Covid-19 infection, with this effect persisting post-recovery (10).
In addition to blood, where cells are already in suspension, the area of applicability has also been extended to tissue biopsy samples. The tissues are dissociated into single-cell suspensions using a tissue-grinder and then analyzed using RT-DC. In this way, inflamed and cancerous colon tissue samples can be reliably distinguished from healthy samples (11).
Recent advances include microfluidic measurements of viscoelastic cell properties. While RT-DC only assesses elastic cell properties, cells also exhibit a time scale for deformation – a fluid-like property (viscosity). To address this, we developed hyperDC – a high-throughput method for viscoelastic measurements in hyperbolic microchannels (12). Additionally, we are exploring shear flow DC (sfDC) and integrating simultaneous fluorescence imaging measurements (13).
To design and produce our microfluidic chips, we work closely with the Lab-on-a-Chip TDSU at MPL.
[1] Urbanska M, Muñoz HE, Shaw Bagnall J, Otto O, Manalis SR, Di Carlo D, et al. A comparison of microfluidic methods for high-throughput cell deformability measurements. Nat Methods. 2020. 17(6):587–593. https://doi.org/10.1038/s41592-020-0818-8
[2] Otto O, Rosendahl P, Mietke A, Golfier S, Herold C, Klaue D, et al. Real-time deformability cytometry: on-the-fly cell mechanical phenotyping. Nat Methods. 2015. 12(3):199–202. https://doi.org/10.1038/nmeth.3281
[3] Urbanska M, Rosendahl P, Kräter M, Guck J. High-throughput single-cell mechanical phenotyping with real-time deformability cytometry. In: Methods in Cell Biology. Academic Press, 2018. p. 175–198. https://doi.org/10.1016/bs.mcb.2018.06.009
[4] Rosendahl P, Plak K, Jacobi A, Kraeter M, Toepfner N, Otto O, et al. Real-time fluorescence and deformability cytometry. Nat Methods. 2018. 15(5):355–358. https://doi.org/10.1038/nmeth.4639
[5] Nawaz AA, Urbanska M, Herbig M, Nötzel M, Kräter M, Rosendahl P, et al. Intelligent image-based deformation-assisted cell sorting with molecular specificity. Nat Method. 2020. 17(6):595–599. https://doi.org/10.1038/s41592-020-0831-y
[6] Mietke A, Otto O, Girardo S, Rosendahl P, Taubenberger A, Golfier S, et al. Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment. Biophys J. 2015. 109(10):2023–2036. https://doi.org/10.1016/j.bpj.2015.09.006
[7] Mokbel M, Mokbel D, Mietke A, Träber N, Girardo S, Otto O, et al. Numerical Simulation of Real-Time Deformability Cytometry To Extract Cell Mechanical Properties. ACS Biomater Sci Eng. 2017. 3(11):2962–2973. https://doi.org/10.1021/acsbiomaterials.6b00558
[8] Wittwer LD, Reichel F, Müller P, Guck J, Aland S. A new hyperelastic lookup table for RT-DC. Soft Matter. 2023. 19(11):2064–2073. https://doi.org/10.1016/j.bpj.2015.09.006
[9] Toepfner N, Herold C, Otto O, Rosendahl P, Jacobi A, Kräter M, et al. Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood. eLife. 2018. 7:e29213. https://doi.org/10.7554/eLife.29213
[9] Kubánková M, Hohberger B, Hoffmanns J, Fürst J, Herrmann M, Guck J, et al. Physical phenotype of blood cells is altered in COVID-19. Biophys J. 2021. 120(14):2838–2847. https://doi.org/10.1016/j.bpj.2021.05.025
[11] Soteriou D, Kubánková M, Schweitzer C, López-Posadas R, Pradhan R, Thoma OM, et al. Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies. Nature Biomedical Engineering. 2023. 7:1392-1403. https://doi.org/10.1038/s41551-023-01015-3
[12] Reichel F, Goswami R, Girardo S, Guck J. High-throughput viscoelastic characterization of cells in hyperbolic microchannels. Lab Chip. 2024. 24(9):2440–2453. https://doi.org/10.1039/D3LC01061A
[13] Gerum R, Mirzahossein E, Eroles M, Elsterer J, Mainka A, Bauer A, et al. Viscoelastic properties of suspended cells measured with shear flow deformation cytometry. eLife. 2022. 11:e78823. https://doi.org/10.7554/eLife.78823
