Coded Illumination for Motion-blur Free Imaging of Cells on Cell-phone Based Imaging Flow Cytometer

Cell-phone based imaging flow cytometry can be realized by flowing cells through the microfluidic devices, and capturing their images with an optically enhanced camera of the cell-phone. Throughput in flow cytometers is usually enhanced by increasing the flow rate of cells. However, maximum frame rate of camera system limits the achievable flow rate. Beyond this, the images become highly blurred due to motion-smear. We propose to address this issue with coded illumination, which enables recovery of high-fidelity images of cells far beyond their motion-blur limit. This paper presents simulation results of deblurring the synthetically generated cell/bead images under such coded illumination.

Indigenous Development of an Imaging Flow Cytometer for Clinical and Biological Applications

Flow cytometry is a benchmark technique used for basic research and clinical diagnosis of various diseases. Despite being a high-throughput technique, it fails in capturing the morphology of cells being analyzed. Imaging flow cytometry is a combination of flow-cytometry and digital microscopy, which offers advantages of both the techniques. In this paper, we report on the development of an indigenous Imaging Flow Cytometer, realized with the combination of Optics, Microfluidics, and High-speed imaging. A custom-made bright-field transmission microscope is used to capture images of cells flowing across the microfluidic device. High-throughput morphological analysis on suspension of yeast cells is presented.

Field-Portable Microfluidics-Based Imaging Flow Cytometer

Clinical microscopy is a versatile and robust tool used for the diagnosis of a plethora of diseases. However, due to various reasons, it remains inaccessible in resource limited settings. In this paper, we present an automated and cost-effective alternative to microscopy for use in clinical diagnostics. With the use of custom optics and microfluidics, we demonstrate a field-portable imaging flow cytometry system. Using the presented system, we have been able to image 586 cells per second. We demonstrate the clinical relevance of the proposed system by differentiating between suspensions of healthy and sphered RBCs based on high-throughput morphometric analysis. The instrument presented here is a major advancement in the domain of field portable diagnostics as it enables fast and robust quantitative diagnostic testing at the point-of-care.

Framework for morphometric classification of cells in imaging flow cytometry

Imaging flow cytometry is an emerging technology that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy. It allows high-throughput imaging of cells with good spatial resolution, while they are in flow. This paper proposes a general framework for the processing/classification of cells imaged using imaging flow cytometer. Each cell is localized by finding an accurate cell contour. Then, features reflecting cell size, circularity and complexity are extracted for the classification using SVM. Unlike the conventional iterative, semi-automatic segmentation algorithms such as active contour, we propose a noniterative, fully automatic graph-based cell localization. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using custom fabricated cost-effective microfluidics-based imaging flow cytometer. The proposed system is a significant development in the direction of building a cost-effective cell analysis platform that would facilitate affordable mass screening camps looking cellular morphology for disease diagnosis. Lay description In this article, we propose a novel framework for processing the raw data generated using microfluidics based imaging flow cytometers. Microfluidics microscopy or microfluidics based imaging flow cytometry (mIFC) is a recent microscopy paradigm, that combines the statistical power of flow cytometry with spatial and quantitative morphology of digital microscopy, which allows us imaging cells while they are in flow. In comparison to the conventional slide-based imaging systems, mIFC is a nascent technology enabling high throughput imaging of cells and is yet to take the form of a clinical diagnostic tool. The proposed framework process the raw data generated by the mIFC systems. The framework incorporates several steps: beginning from pre-processing of the raw video frames to enhance the contents of the cell, localising the cell by a novel, fully automatic, non-iterative graph based algorithm, extraction of different quantitative morphological parameters and subsequent classification of cells. In order to evaluate the performance of the proposed framework, we have successfully classified unstained label-free leukaemia cell-lines MOLT, K562 and HL60 from video streams captured using cost-effective microfluidics based imaging flow cytometer. The cell lines of HL60, K562 and MOLT were obtained from ATCC (American Type Culture Collection) and are separately cultured in the lab. Thus, each culture contains cells from its own category alone and thereby provides the ground truth. Each cell is localised by finding a closed cell contour by defining a directed, weighted graph from the Canny edge images of the cell such that the closed contour lies along the shortest weighted path surrounding the centroid of the cell from a starting point on a good curve segment to an immediate endpoint. Once the cell is localised, morphological features reflecting size, shape and complexity of the cells are extracted and used to develop a support vector machine based classification system. We could classify the cell-lines with good accuracy and the results were quite consistent across different cross validation experiments. We hope that imaging flow cytometers equipped with the proposed framework for image processing would enable cost-effective, automated and reliable disease screening in over-loaded facilities, which cannot afford to hire skilled personnel in large numbers. Such platforms would potentially facilitate screening camps in low income group countries; thereby transforming the current health care paradigms by enabling rapid, automated diagnosis for diseases like cancer.

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.

Unsteady flow and heat transfer between rotating coaxial disks

A study is made on the flow and heat transfer of a viscous fluid confined between two parallel disks. The disks are allowed to rotate with different time dependent angular velocities, and the upper disk is made to approach the lower one with a constant speed. Numerical solutions of the governing parabolic partial differential equations are obtained through a fourth-order accurate compact finite difference scheme. The normal forces and torques that the fluid exerts on the rotating surfaces are obtained at different nondimensional times for different values of the rate of squeezing and disk angular velocities. The temperature distribution and heat transfer are also investigated in the present analysis.

Unsteady Compressible Boundary-Layer Flow On The Stagnation Line Of An Infinite Swept Cylinder

Numerical solutions of flow and heat transfer process on the unsteady flow of a compressible viscous fluid with variable gas properties in the vicinity of the stagnation line of an infinite swept cylinder are presented. Results are given for the case where the unsteady temperature field is produced by (i) a sudden change in the wall temperature (enthalpy) as the impulsive motion is started and (ii) a sudden change in the free-stream velocity. Solutions for the simultaneous development of the thermal and momentum boundary layers are obtained by using quasilinearization technique with an implicit finite difference scheme. Attention is given to the transient phenomenon from the initial flow to the final steady-state distribution. Results are presented for the skin friction and heat transfer coefficients as well as for the velocity and enthalpy profiles. The effects of wail enthalpy parameter, sweep parameter, fluid properties and transpiration cooling on the heat transfer and skin friction are considered.

Unsteady-Flow Of A Viscous-Fluid Between 2 Parallel Disks With A Time-Varying Gap Width And A Magnetic-Field

The unsteady incompressible viscous fluid flow between two parallel infinite disks which are located at a distance h(t*) at time t* has been studied. The upper disk moves towards the lower disk with velocity h'(t*). The lower disk is porous and rotates with angular velocity Omega(t*). A magnetic field B(t*) is applied perpendicular to the two disks. It has been found that the governing Navier-Stokes equations reduce to a set of ordinary differential equations if h(t*), a(t*) and B(t*) vary with time t* in a particular manner, i.e. h(t*) = H(1 - alpha t*)(1/2), Omega(t*) = Omega(0)(1 - alpha t*)(-1), B(t*) = B-0(1 - alpha t*)(-1/2). These ordinary differential equations have been solved numerically using a shooting method. For small Reynolds numbers, analytical solutions have been obtained using a regular perturbation technique. The effects of squeeze Reynolds numbers, Hartmann number and rotation of the disk on the flow pattern, normal force or load and torque have been studied in detail

A water model study of the flow asymmetry inside a continuous slab casting mold

The work studies the extent of asymmetric flow in water models of continuous casting molds of two different configurations. In the molds where fluid is discharged through multiple holes at the bottom, the flow pattern in the lower portion depends on the size of the lower two recirculating domains. If they reach the mold bottom, the flow pattern in the lower portion is symmetrical about the central plane; otherwise, it is asymmetrical. On the other hand, in the molds where the fluid is discharged through the entire mold cross section, the flow pattern is always asymmetrical if the aspect ratio is 1:6.25 or more. The fluid jet swirls while emerging through the nozzle. The interaction of the swirling Jets with the wide sidewalls of the mold gives rise to asymmetrical flow inside the mold. In the molds with lower aspect ratios, where the jets do not touch the wide side walls, the flow pattern is symmetrical about the central plane.

Unsteady flow over a stretching surface with a magnetic field in a rotating fluid

The effect of the magnetic field on the unsteady flow over a stretching surface in a rotating fluid has been studied. The unsteadiness in the flow field is due to the time-dependent variation of the velocity of the stretching surface and the angular velocity of the rotating fluid. The Navier-Stokes equations and the energy equation governing the flow and the heat transfer admit a self-similar solution if the velocity of the stretching surface and the angular velocity of the rotating fluid vary inversely as a linear function of time. The resulting system of ordinary differential equations is solved numerically using a shooting method. The rotation parameter causes flow reversal in the component of the velocity parallel to the strerching surface and the magnetic field tends to prevent or delay the flow reversal. The surface shear stresses dong the stretching surface and in the rotating direction increase with the rotation parameter, but the surface heat transfer decreases. On the other hand, the magnetic field increases the surface shear stress along the stretching surface, but reduces the surface shear stress in the rotating direction and the surface heat transfer. The effect of the unsteady parameter is more pronounced on the velocity profiles in the rotating direction and temperature profiles.

Unsteady free convection flow in the stagnation-point region of a rotating sphere

The unsteady free convection boundary-layer flow in the forward stagnation-point region of a sphere, which is rotating with time-dependent angular velocity in an ambient fluid, has been studied. Both constant wall temperature and constant hear flux conditions have been considered. The non-linear coupled parabolic partial differential equations governing the flow have been solved numerically using an implicit finite-difference scheme. The skin friction and the heat transfer are enhanced by the buoyancy force. The effect of the buoyancy force is found to be more pronounced for smaller Prandtl numbers than for larger Prandtl numbers. For a given buoyancy force, the heat transfer increases with an increase in Prandtl number, but the skin friction decreases.

Unsteady free convection flow in the stagnation-point region of a three-dimensional body

The unsteady free convection flow in the stagnation-point region of a heated three-dimensional body placed in an ambient fluid is studied under boundary layer approximations. We have considered the case where there is an initial steady state that is perturbed by a step-change in the wall temperature. The non-linear coupled partial differential equations governing the free convection flow are solved numerically using a finite difference scheme. The presented results show the temporal development of the momentum and thermal boundary layer characteristics.

A Ca2+-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry

When freshly starved amoebae of Dictyostelium discoideum are loaded with the Ca2+-specific dye indo-1/AM and analyzed in a fluorescence-activated cell sorter, they exhibit a quasi-bimodal distribution of fluorescence. This permits a separation of the population into two classes: H, or ''high Ca2+-indo-1 fluorescence,'' and L, or ''low Ca2+-indo-1 fluorescence.'' Simultaneous monitoring of Ca2+-indo-1 and Ca2+-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca2+. Next we label H cells with tetramethylrhodamine isothiocyanate (TRITC) and mix them in a 1:4 ratio with L cells, In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca2+ at the vegetative stage tend to develop into prestalk cells and those with low Ca2+ into prespores. Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca2+-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azhar ef al., Curr. Sci. 68, 337-342 (1995)), a prestalk-prespore difference in cellular Ca2+ is present in the cells of the slug in vivo. These findings are discussed in light of the possible roles of Ca2+ for cell differentiation in D. discoideum.

Mixed Convection Boundary Layer Flow Past a Horizontal Circular Cylinder Embedded in a Bidisperse Porous Medium

Adopting a two-temperature and two-velocity model, appropriate to a bidisperse porous medium (BDPM) proposed by Nield and Kuznetsov (2008), the classical steady, mixed convection boundary layer flow about a horizontal, isothermal circular cylinder embedded in a porous medium has been theoretically studied in this article. It is shown that the boundary layer analysis leads to expressions for the flow and heat transfer characteristics in terms of an inter-phase momentum parameter, a thermal diffusivity ratio, a thermal conductivity ratio, a permeability ratio, a modified thermal capacity ratio, and a buoyancy or mixed convection parameter. The transformed partial differential equations governing the flow and heat transfer in the f-phase (the macro-pores) and the p-phase (the remainder of the structure) are solved numerically using a very efficient implicit finite-difference technique known as Keller-box method. A good agreement is observed between the present results and those known from the open literature in the special case of a traditional Darcy formulation (monodisperse system).

Fast decay of the velocity autocorrelation function in dense shear flow of inelastic hard spheres

We find in complementary experiments and event-driven simulations of sheared inelastic hard spheres that the velocity autocorrelation function psi(t) decays much faster than t(-3/2) obtained for a fluid of elastic spheres at equilibrium. Particle displacements are measured in experiments inside a gravity-driven flow sheared by a rough wall. The average packing fraction obtained in the experiments is 0.59, and the packing fraction in the simulations is varied between 0.5 and 0.59. The motion is observed to be diffusive over long times except in experiments where there is layering of particles parallel to boundaries, and diffusion is inhibited between layers. Regardless, a rapid decay of psi(t) is observed, indicating that this is a feature of the sheared dissipative fluid, and is independent of the details of the relative particle arrangements. An important implication of our study is that the non-analytic contribution to the shear stress may not be present in a sheared inelastic fluid, leading to a wider range of applicability of kinetic theory approaches to dense granular matter.