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Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells—small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations. 

Dielectrophoresis (DEP) is a powerful tool for label-free sorting of cells, even those with subtle differences in morphological and dielectric properties. Nevertheless, a major limitation is that most existing DEP techniques can efficiently sort cells only at low throughputs (<1 mL h−1). Here, we demonstrate that the integration of a three-dimensional (3D) coupled hydrodynamic-DEP cell pre-focusing module upstream of the main DEP sorting region enables cell sorting with a 10-fold increase in throughput compared to conventional DEP approaches. To better understand the key principles and requirements for high-throughput cell separation, we present a comprehensive theoretical model to study the scaling of hydrodynamic and electrostatic forces on cells at high flow rate regimes. Based on the model, we show that the critical cell-to-electrode distance needs to be ≤10 µm for efficient cell sorting in our proposed microfluidic platform, especially at flow rates ≥ 1 mL h−1. Based on those findings, a computational fluid dynamics model and particle tracking analysis were developed to find optimum operation parameters (e.g., flow rate ratios and electric fields) of the coupled hydrodynamic-DEP 3D focusing module. Using these optimum parameters, we experimentally demonstrate live/dead K562 cell sorting at rates as high as 10 mL h−1 (>150,000 cells min−1) with 90% separation purity, 85% cell recovery, and no negative impact on cell viability. 

We present an integrated microfluidic chip capable of label-free isolation of three major subpopulations of white blood cells (WBCs) (lymphocytes, monocytes and granulocytes) from undiluted whole blood. The proposed system accomplishes 3-part differential sorting of WBCs by: (1) On-chip lysis of RBCs from the blood sample, and (2) Downstream isolation of lymphocytes, monocytes and granulocytes using dielectrophoresis (DEP) technology. 

Insulator-based microfluidic devices are attractive for handling biological samples due to their simple fabrication, low-cost, and efficiency in particle manipulation. However, their widespread application is limited by the high operation voltages required to achieve particle trapping. We present a theoretical, numerical, and experimental study that demonstrates these voltages can be significantly reduced (to sub-100 V) in direct-current insulator-based electrokinetic (DC-iEK) devices for micron-sized particles. To achieve this, we introduce the concept of the amplification factor—the fold-increase in electric field magnitude due to the presence of an insulator constriction—and use it to compare the performance of different microchannel designs and to direct our design optimization process. To illustrate the effect of using constrictions with smooth and sharp features on the amplification factor, geometries with circular posts and semi-triangular posts were used. These were theoretically approximated in two different systems of coordinates (bipolar and elliptic), allowing us to provide, for the first time, explicit electric field amplification scaling laws. Finite element simulations were performed to approximate the 3D insulator geometries and provide a parametric study of the effect of changing different geometrical features. These simulations were used to predict particle trapping voltages for four different single-layer microfluidic devices using two particle suspensions (2 and 6.8 μm in size). The general agreement between our models demonstrates the feasibility of using the amplification factor, in combination with nonlinear electrokinetic theory, to meet the prerequisites for the development of portable DC-iEK microfluidic systems. 

Abstract Insulator‐based dielectrophoresis (iDEP) is the electrokinetic migration of polarized particles when subjected to a non‐uniform electric field generated by the inclusion of insulating structures between two remote electrodes. Electrode spacing is considerable in iDEP systems when compared to electrode‐based DEP systems, therefore, iDEP systems require high voltages to achieve efficient particle manipulation. A consequence of this is the temperature increase within the channel due to Joule heating effects, which, in some cases, can be detrimental when manipulating biological samples. This work presents an experimental and modeling study on the increase in temperature inside iDEP devices. For this, we studied seven distinct channel designs that mainly differ from each other in their post array characteristics: post shape, post size and spacing between posts. Experimental results obtained using a custom‐built copper Resistance Temperature Detector, based on resistance changes, show that the influence of the insulators produces a difference in temperature rise of approximately 4°C between the designs studied. Furthermore, a 3D COMSOL model is also introduced to evaluate heat generation and dissipation, which is in good agreement with the experiments. The model allowed relating the difference in average temperature for the geometries under study to the electric resistance posed by the post array in each design.