Label-free liquid biopsy through the identification of tumor cells by machine learning-powered tomographic phase imaging flow cytometry.

Image-based identification of circulating tumor cells in microfluidic cytometry condition is one of the most challenging perspectives in the Liquid Biopsy scenario. Here we show a machine learning-powered tomographic phase imaging flow cytometry system capable to provide high-throughput 3D phase-contrast tomograms of each single cell. In fact, we show that discrimination of tumor cells against white blood cells is potentially achievable with the aid of artificial intelligence in a label-free flow-cyto-tomography method. We propose a hierarchical machine learning decision-maker, working on a set of features calculated from the 3D tomograms of the cells' refractive index. We prove that 3D morphological features are adequately distinctive to identify tumor cells versus the white blood cell background in the first stage and, moreover, in recognizing the tumor type at the second decision step. Proof-of-concept experiments are shown, in which two different tumor cell lines, namely neuroblastoma cancer cells and ovarian cancer cells, are used against monocytes. The reported results allow claiming the identification of tumor cells with a success rate higher than 97% and with an accuracy over 97% in discriminating between the two cancer cell types, thus opening in a near future the route to a new Liquid Biopsy tool for detecting and classifying circulating tumor cells in blood by stain-free method.

Dynamics of prolate spheroids in the vicinity of an air-water interface.

In this article, we present the mobilities of prolate ellipsoidal micrometric particles close to an air-water interface measured by dual wave reflection interference microscopy. Particle's position and orientation with respect to the interface are simultaneously measured as a function of time. From the measured mean square displacement, five particle mobilities (3 translational and 2 rotational) and two translational-rotational cross-correlations are extracted. The fluid dynamics governing equations are solved by the finite element method to numerically evaluate the same mobilities, imposing either slip and no-slip boundary conditions to the flow at the air-water interface. The comparison between experiments and simulations reveals an agreement with no-slip boundary conditions prediction for the translation normal to the interface and the out-of-plane rotation, and with slip ones for parallel translations and in-plane rotation. We rationalize these evidences in the framework of surface incompressibility at the interface.

An Experimental and Numerical Investigation on Bubble Growth in Polymeric Foams.

The cellular morphology of thermoplastic polymeric foams is a key factor for their performances. Three possible foam morphologies exist, namely, with closed cells, interconnected cellular structure, and open cells. In the gas foaming technology, a physical blowing agent, e.g., CO2 or N2, is used to form bubbles at high pressure in softened/melted polymers. As a consequence of a pressure quench, the bubbles grow in the liquid matrix until they impinge and possibly break the thin liquid films among them. If film breakage happens, the broken film may retract due to the elastic energy accumulated by the polymeric liquid during the bubble growth. This, in turn, determines the final morphology of the foam. In this work, we experimentally study the growth of CO2 bubbles in a poly(e-caprolactone) (PCL) matrix under different pressure conditions. In addition, we perform three-dimensional direct numerical simulations to support the experimental findings and rationalize the effects of the process parameters on the elastic energy accumulated in the liquid at the end of the bubble growth, and thus on the expected morphology of the foam. To do that, we also extend the analytic model available in the literature for the growth of a single bubble in a liquid to the case of a liquid with a multi-mode viscoelastic constitutive equation.

Axisymmetric bare freestanding films of highly viscous liquids: Preparation and real-time investigation of capillary leveling.

HYPOTHESIS: Thin liquid films are important in many scientific fields. In particular, films with both the surface layers exposed to a different fluid phase, known as freestanding films, are relevant in the ambit of foams and emulsions. Hence, there is a great interest in developing novel techniques allowing to form large and stable freestanding liquid films and to follow their dynamics. EXPERIMENTS: We develop a novel opto-mechanical tool allowing to perform and study the preparation and the capillary leveling flow of axisymmetric bare freestanding liquid films. The tool is composed by a customized motorized iris diaphragm and by an innovative joint imaging setup combining digital holography and white light color interferometry that enables real-time measurement of film thickness over a large field of view. The dynamics of films made of a model Newtonian fluid, i.e., high-viscosity silicone oil, is studied. Direct numerical simulations and a hydrodynamic model based on the lubrication theory are used to support the experimental results. FINDINGS: Iris opening induces the formation of large circular freestanding films with a stepped profile. Once iris opening is stopped, the films undergo a capillary leveling flow tending to flatten their profile. The leveling flow follows the theoretical scaling given by Ilton et al. [1]. We prove through numerical simulations that an equi-biaxial extensional flow occurs at the film center. Furthermore, we observe the formation and dynamics of dimples in bare freestanding films for the first time.

Dissolution of concentrated surfactant solutions: from microscopy imaging to rheological measurements through numerical simulations.

Concentrated aqueous solutions of surfactants, often referred to as pastes, experience complex phase and rheology changes upon dissolution in water, which is a typical step in the production of liquid detergents. During the dilution process, depending on water content, surfactant molecules can arrange in different morphologies, such as lamellar or cubic and hexagonal structures. These phases are characterized by different physico-chemical properties, such as viscosity or diffusivity, which lead to non-simple transport mechanisms during the dissolution process. In this work, we investigate the dissolution of concentrated Sodium Lauryl Ether Sulfate (SLES) pastes in water under quiescent conditions by coupling different experimental techniques. A thorough rheological characterization of the system showed non-monotonic changes of several orders of magnitude in its viscosity and viscoelastic moduli as a function of water content. Time-lapse microscopy allowed us to image the dynamic evolution of the phase changes as water penetrated in a disk-shaped sample (with the same geometry used in rheological tests). Numerical simulation, based on a simple diffusion-based multi-parameter model is shown to describe satisfactorily SLES dissolution data.

Assembling and rotating erythrocyte aggregates by acoustofluidic pressure enabling full phase-contrast tomography.

The combined use of ultrasound radiation and microfluidics is a promising tool for aiding the development of lab-on-a-chip devices. In this study, we show that the rotation of linear aggregates of micro-particles can be achieved under the action of acoustic field pressure. This novel manipulation is investigated by tracking polystyrene beads of different sizes through the 3D imaging features of digital holography (DH). From our analysis it is understood that the positioning of the micro-particles and their aggregations are associated with the effect of bulk acoustic radiation forces. The observed rotation is instead found to be compatible with the presence of acoustic streaming patterns as evidenced by our modelling and the resulting numerical simulation. Furthermore, the rotation frequency is shown to depend on the input voltage applied on the acoustic device. Finally, we demonstrate that we can take full advantage of such rotation by combining it with quantitative phase imaging of DH for a significant lab-on-a-chip biomedical application. In fact, we demonstrate that it is possible to put in rotation a linear aggregate of erythrocytes and rely on holographic imaging to achieve a full phase-contrast tomography of the aforementioned aggregate.

Design of a microfluidic device for the measurement of the elastic modulus of deformable particles.

A microfluidic technique recently proposed in the literature to measure the interfacial tension between a liquid droplet and an immiscible suspending liquid [Hudson et al., Appl. Phys. Lett., 2005, 87, 081905], [Cabral and Hudson, Lab Chip, 2006, 6, 427] is suitably adapted to the characterization of the elastic modulus of soft particles in a continuous-flow process. A microfluidic device consisting of a cylindrical pipe with a reduction in cross-section is designed, and the deformation and velocity of incompressible elastic particles suspended in a Newtonian liquid are tracked as they move along the centerline through the constriction. Kinematic and shape information is exploited to calculate the particle's elastic modulus by means of the theory of elastic particle deformation in extensional flow. The approach is validated for different orders of magnitude of the elastic capillary number through experiments and numerical simulations.

Full-angle tomographic phase microscopy of flowing quasi-spherical cells.

We report a reliable full-angle tomographic phase microscopy (FA-TPM) method for flowing quasi-spherical cells along microfluidic channels. This method lies in a completely passive optical system, i.e. mechanical scanning or multi-direction probing of the sample is avoided. It exploits the engineered rolling of cells while they are flowing along a microfluidic channel. Here we demonstrate significant progress with respect to the state of the art of in-flow TPM by showing a general extension to cells having almost spherical shapes while they are flowing in suspension. In fact, the adopted strategy allows the accurate retrieval of rotation angles through a theoretical model of the cells' rotation in a dynamic microfluidic flow by matching it with phase-contrast images resulting from holographic reconstructions. So far, the proposed method is the first and the only one that permits to get in-flow TPM by probing the cells with full-angle, achieving accurate 3D refractive index mapping and the simplest optical setup, simultaneously. Proof of concept experiments were performed successfully on human breast adenocarcinoma MCF-7 cells, opening the way for the full characterization of circulating tumor cells (CTCs) in the new paradigm of liquid biopsy.

Magnetophoresis 'meets' viscoelasticity: deterministic separation of magnetic particles in a modular microfluidic device.

The deflection of magnetic beads in a microfluidic channel through magnetophoresis can be improved if the particles are somehow focused along the same streamline in the device. We design and fabricate a microfluidic device made of two modules, each one performing a unit operation. A suspension of magnetic beads in a viscoelastic medium is fed to the first module, which is a straight rectangular-shaped channel. Here, the magnetic particles are focused by exploiting fluid viscoelasticity. Such a channel is one inlet of the second module, which is a H-shaped channel, where a buffer stream is injected in the second inlet. A permanent magnet is used to displace the magnetic beads from the original to the buffer stream. Experiments with a Newtonian suspending fluid, where no focusing occurs, are carried out for comparison. When viscoelastic focusing and magnetophoresis are combined, magnetic particles can be deterministically separated from the original streamflow to the buffer, thus leading to a high deflection efficiency (up to ~96%) in a wide range of flow rates. The effect of the focusing length on the deflection of particles is also investigated. Finally, the proposed modular device is tested to separate magnetic and non-magnetic beads.

Single line particle focusing induced by viscoelasticity of the suspending liquid: theory, experiments and simulations to design a micropipe flow-focuser.

We perform 3D numerical simulations, heuristic modeling and microfluidic experiments to demonstrate, for the first time, the presence of a bistability scenario for transversal migration of particles suspended in a viscoelastic liquid flowing in a pipe. Our results show that particle migration, either at the centerline or at the wall, can be controlled by the rheological properties of the suspending liquid and by the relative dimensions of the particle and tube. Proper selection of these parameters can promote strict aligning of particles on a line, i.e., 3-D focusing. Simple design rules are given to rationally control particle focusing under flow in micropipes.