Label-free single cell analysis methods are of growing interest because they provide direct measurement of phenotype, particularly mechanical or electrical properties. The mechanical properties manifest through cell deformability are closely related to intracellular structure, particularly of the cytoskeleton and nucleus1. Many different properties are linked to cell deformation, including cell cycle2,3, cancer4,5,6 immune cell activation7,8 and stem cell differentiation6,9. Single cell mechanical phenotyping can be performed direct from biopsy samples in order to determine inflammation and discriminate healthy from tumour tissue10.
Single-cell mechanical analysis is performed using several different techniques11, including AFM12,13, acoustic scattering14, optical stretching15, and micropipette aspiration16. However, these methods are not high throughput and can be technically demanding, and to address this microfluidic single cell cytometric methods have been developed1. One technique is contact-based deformability cytometry (cDC), where cell stiffness is determined from the transit time as it squeezes through a narrow constriction. The transit time is measured using techniques such as optical imaging17,18,19 resonating cantilever methods (which can also determine cell buoyant mass)20, or electrical resistance/impedance methods21,22,23,24, including electrical node pore sensing25. Constriction based methods have also been developed to characterise both the electrical and mechanical properties of single cells26. However, contact based methods are generally low throughput, influenced by clogging of the channel and measure a narrow range of cell sizes. Furthermore, cell transit time is influenced not only by deformability but also by cell volume and membrane-wall friction and interactions.
To address these limitations non-contact analysis methods have been where a hydrodynamic flow induces a shape change in the cell, eliminating interaction between the cell and the channel wall. High speed cameras and image processing measure cell shape from which cell deformability is inferred. Shear flow deformability cytometry (sDC) uses strong velocity gradients to generate shear stress in a microchannel slightly larger than the cell to deform the cell into a bullet shape27,28. Shear forces dominate, and this technique is mostly sensitive to changes in the cytoskeleton but not the nuclear structure.
Extensional flow deformability cytometry (xDC) uses fluid-induced stress to deform cells at a stagnation point, normally with a cross shaped microfluidic channel5,6,29. Inertial forces induce changes in a few micro-seconds meaning that analysis rates exceed 1,000 cells per second (high Re). The dominant compressional force from the fluid inertia deforms the cells. Guillou et al30 used an extensional flow device but at much lower Re numbers where shear forces dominate and observed changes due to actin destabilisation. Armistead et al31 described a device that covers both flow regimes from high to low strain in both shear and inertia dominant regimes and showed that different regimes probe different aspects of the cell structure, demonstrating that the shear-dominant, low-strain regime is most sensitive to cytoskeletal changes. The three different techniques were recently compared32, confirming that the higher strain rate of xDC makes measurement of cytoskeletal changes (actin destabilisation) challenging, possibly due to cytoskeletal fluidization31.
Analogous to the field of cell mechanics, probing cell phenotypic electrical properties has been of interest for many years. Traditionally cells were analysed in suspension, but microfluidic technologies enable high speed single cells, allow heterogeneity in populations to be identified. Cell electrical properties reflect fundamental cellular physiology, for example cell cycle33, activation/function34, cytoskeleton35; and single cell impedance analysis has been used for tumour cell stratification/separation36,37, leukocyte analysis38 and to identify parasite invasion39. Single cell impedance analysis is usually performed using microfluidic devices with micro-electrodes that measure the impedance of a microchannel as cells transit between successive pairs of electrodes40,41. Traditionally measurements are made at two AC frequencies, typically a lower frequency (high kHz) to measure cell volume and a second higher frequency to measure cell membrane properties. The ratio of these two impedances is termed the electrical opacity41 and indirectly characterises the cell membrane. Single cell multi-frequency measurements have also been demonstrated providing a complete electrical phenotype by fitting data to a lumped-parameter model42.
Given the growing interest in label free techniques, and their translational potential for diagnosing disease, techniques that simultaneously measure both the mechanical and electrical properties of cells may provide important insights into cell behaviour and disease pathology. Recently a non-contact impedance-based deformability cytometer was described43. This system measures cell deformability using electrical rather than optical methods and measures both the electrical and mechanical properties of single cells at moderate throughput (10–20 cells per second). Viscoelastic-inertial sheath flow is used to focus cells into a narrow stream that flows through a cross-junction where cells are deformed due to pinching from sheath fluids. In this system shear force dominates over the compressive force. The change in cell shape was determined by comparing the impedance signal before and after a cell passes along the cross-shaped microchannel. Size, deformability and electrical opacity of neutrophils was measured, demonstrating changes upon activation. Reale et al44 used extensional flow created with a hyperbolic channel to induce cell deformation. Planar microelectrodes at a cross junction measure the orthogonal and lateral impedance to determine cell shape after deformation. Differences between normal RBCs and stiffer spherical RBCs (treated with SDS and Glutaraldehyde) were identified. Owing to variations in the electrical impedance signal with position in the channel, off-centre particles were discarded (based on velocity), corresponding to around 50% of total events.
In this paper we describe a high throughput single cell shear flow deformability cytometer (sDC) that simultaneously measures the mechanical and electrical properties of single cells at high throughput (> 100s− 1). The method is simple and does not require a separate sheath flow or high-speed cameras with associated data processing overheads. Cells suspended in a viscoelastic buffer and are pumped through a narrow channel, producing a shear force to induce cell deformation whilst also focusing cells into the channel centre46. Cell deformability and electrical properties are measured using integrated planar microelectrodes, at two discrete frequencies giving cell volume, shape and cell electrical properties. As a cell flows through a channel the electrical impedance is measured along two orthogonal axes to determine any change in the cell as it deforms (from sphere to ellipse) along with the electrical volume and opacity.
We demonstrate the utility of this technique by measuring the combined mechanical and electrical properties of HL60 cells under several different experimental conditions, including osmotic stress, Glutaraldehyde (GA) cross-linking, and cytoskeleton disruption. This new electro-mechanical phenotyping is simple and inexpensive. The system is high throughput, simple and does not require complex fluidics or sheath flow focusing and provides equivalent data to other optical deformability cytometry methods.