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.

Effects of refractive index mismatch in optical CT imaging of polymer gel dosimeters

Purpose: Proposing an image reconstruction technique, algebraic reconstruction technique-refraction correction (ART-rc). The proposed method takes care of refractive index mismatches present in gel dosimeter scanner at the boundary, and also corrects for the interior ray refraction. Polymer gel dosimeters with high dose regions have higher refractive index and optical density compared to the background medium, these changes in refractive index at high dose results in interior ray bending. Methods: The inclusion of the effects of refraction is an important step in reconstruction of optical density in gel dosimeters. The proposed ray tracing algorithm models the interior multiple refraction at the inhomogeneities. Jacob's ray tracing algorithm has been modified to calculate the pathlengths of the ray that traverses through the higher dose regions. The algorithm computes the length of the ray in each pixel along its path and is used as the weight matrix. Algebraic reconstruction technique and pixel based reconstruction algorithms are used for solving the reconstruction problem. The proposed method is tested with numerical phantoms for various noise levels. The experimental dosimetric results are also presented. Results: The results show that the proposed scheme ART-rc is able to reconstruct optical density inside the dosimeter better than the results obtained using filtered backprojection and conventional algebraic reconstruction approaches. The quantitative improvement using ART-rc is evaluated using gamma-index. The refraction errors due to regions of different refractive indices are discussed. The effects of modeling of interior refraction in the dose region are presented. Conclusions: The errors propagated due to multiple refraction effects have been modeled and the improvements in reconstruction using proposed model is presented. The refractive index of the dosimeter has a mismatch with the surrounding medium (for dry air or water scanning). The algorithm reconstructs the dose profiles by estimating refractive indices of multiple inhomogeneities having different refractive indices and optical densities embedded in the dosimeter. This is achieved by tracking the path of the ray that traverses through the dosimeter. Extensive simulation studies have been carried out and results are found to be matching that of experimental results. (C) 2015 American Association of Physicists in Medicine.

Remarkable Selectivity and Photo-Cytotoxicity of an Oxidovanadium(IV) Complex of Curcumin in Visible Light

Oxidovanadium(IV) complexes of 2-(2'-pyridyl)-1,10-phenanthroline (pyphen), viz. VO(pyphen)(acac)](ClO4) (1), VO(pyphen)(anacac)](ClO4) (2) and VO(pyphen)(cur)](ClO4) (3), where acac is acetylacetonate (in 1), anacac is anthracenylacetylacetonate (in 2) and cur is curcumin monoanion (in 3) were synthesized, characterized and their photo-induced DNA cleavage activities and photo-cytotoxicities studied. The complexes are 1: 1 electrolytes in DMF. The one-electron paramagnetic complexes show a d-d band near 760 nm in DMF. Complexes 2 and 3 are blue and green emissive, respectively, in DMSO. The complexes exhibit irreversible V-IV/V-III reductive responses near -1.1 V and V-V/V-IV oxidative responses near 0.85 V vs. SCE in DMF-0.1 M TBAP. Complexes 2 and 3 display significant and selective photo-cytotoxicity upon irradiation with visible light giving an IC50 value of about 5 mu M against HeLa and MCF-7 cancer cells; they are significantly less-toxic against normal 3T3 control cells and in the absence of light. Complex 1 was used as a control. Both cytosolic and nuclear localization of the complexes were observed on the basis of fluorescence imaging. The complexes, avid binders to calf thymus (ct) DNA, were found to photocleave supercoiled pUC19 DNA upon irradiation with near-IR light (785 nm) by generating hydroxyl radical (OH) as the reactive oxygen species (ROS). Cell death events noted with HeLa and MCF-7 cell lines likely are attributable to apoptotic pathways involving light-assisted generation of intracellular ROS.

Remarkable visible light-triggered cytotoxicity of mitochondria targeting mixed-ligand cobalt(III) complexes of curcumin and phenanthroline bases binding to human serum albumin

Six new mixed-ligand cobalt(III) complexes of formulation Co(N-N)(2)(O-O)](ClO4)(2) (1-6), where N-N is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 2), dipyrido3,2-d:2',3'-f] quinoxaline (dpq in 3, 4), and dipyrido3,2-a:2',3'-c]phenazine (dppz in 5, 6), O-O is acetylacetonate (acac in 1, 3, 5) or curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, cur in 2, 4, 6), have been synthesized and characterized. The X-ray crystal structures of complex 1 (as PF6- salt, 1a) and 3 show distorted octahedral geometries formed by the CoN4O2 core. The complexes 1, 3 and 5 having the simple acac ligand are prepared as control species to understand the role of curcumin. The optimized geometries and the frontier orbitals of the curcumin complexes 2, 4, and 6 are obtained from the DFT calculations. The complexes 2, 4, and 6 having the photoactive curcumin moiety display an absorption band in the visible region near 420 nm and show remarkable photocytotoxicity in HeLa cancer cells with respective IC50 values of 7.4 mu M, 5.1 mu M and 1.6 mu M while being much less toxic in dark. MTT assay using complex 6 shows that it is not significantly photocytotoxic to MCF-10A normal cells. The control complexes having the acac ligand are non-toxic both in the presence and absence of light. The cell death is apoptotic in nature and triggered by the photogeneration of reactive oxygen species. Fluorescence imaging experiments on HeLa cells reveals that complex 6 accumulated primarily inside the mitochondria. Human serum albumin (HSA) binding experiments show that the complexes bind HSA with good affinity, but 6 binds with the highest affinity, with a K-b value of 9.8 x 10(5) M-1. Thus, complex 6 with its negligible toxicity in the dark and in normal cells but remarkable toxicity in visible light holds significant photochemotherapeutic potential.

3D Macroporous Solids from Chemically Cross-linked Carbon Nanotubes

We demonstrate a new technique to generate multiple light-sheets for fluorescence microscopy. This is possible by illuminating the cylindrical lens using multiple copies of Gaussian beams. A diffraction grating placed just before the cylindrical lens splits the incident Gaussian beam into multiple beams traveling at different angles. Subsequently, this gives rise to diffraction-limited light-sheets after the Gaussian beams pass through the combined cylindrical lens-objective sub-system. Direct measurement of field at and around the focus of objective lens shows multi-sheet pattern with an average thickness of 7.5 μm and inter-sheet separation of 380 μm. Employing an independent orthogonal detection sub-system, we successfully imaged fluorescently-coated yeast cells (≈4 μm) encaged in agarose gel-matrix. Such a diffraction-limited sheet-pattern equipped with dedicated detection system may find immediate applications in the field of optical microscopy and fluorescence imaging. © 2015 Optical Society of America

Al3+-Ion-Triggered Conformational Isomerization of a Rhodamine B Derivative Evidenced by a Fluorescence Signal - A Crystallographic Proof

A newly designed rhodamine B anisaldehyde hydrazone exhibits Al3+-ion-induced cis (L) to trans (L) conformational isomerization with respect to the xanthene moiety through a rotation about a N-N bond; the isomerization is indicated by a detectable naked-eye color change and a turn-on red fluorescence in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (EtOH/Water 1:9 v/v; pH 7.4) at 25 degrees C. In support of this observation, detailed spectroscopic and physicochemical studies along with density function theory (DFT) calculations have been performed. This cis-to-trans conformational isomerization is due to Al3+ ion coordination, which induces this visual color change and the turn-on fluorescence response. To strengthen our knowledge of the conformational isomerization, detailed structural characterizations of the cis and trans isomers in the solid state were performed by single-crystal X-ray diffraction. To the best of our knowledge, this is the first structural report of both cis and trans conformational isomers for this family of compounds. Moreover, this noncytotoxic probe could be used to image the accumulation of Al3+ ions in HeLa and MCF-7 cell lines.

A Gold Sensors Array for Imaging The Real Tissue Phantom in Electrical Impedance Tomography

Surface electrodes in Electrical Impedance Tomography (EIT) phantoms usually reduce the SNR of the boundary potential data due to their design and development errors. A novel gold sensors array with high geometric precision is developed for EIT phantoms to improve the resistivity image quality. Gold thin films are deposited on a flexible FR4 sheet using electro-deposition process to make a sixteen electrode array with electrodes of identical geometry. A real tissue gold electrode phantom is developed with chicken tissue paste and the fat cylinders as the inhomogeneity. Boundary data are collected using a USB based high speed data acquisition system in a LabVIEW platform for different inhomogeneity positions. Resistivity images are reconstructed using EIDORS and compared with identical stainless steel electrode systems. Image contrast parameters are calculated from the resistivity matrix and the reconstructed images are evaluated for both the phantoms. Image contrast and image resolution of resistivity images are improved with gold electrode array.

Graphene oxide-Fe3O4 nanoparticle composite with high transverse proton relaxivity value for magnetic resonance imaging

The potential of graphene oxide-Fe3O4 nanoparticle (GO-Fe3O4) composite as an image contrast enhancing material in magnetic resonance imaging has been investigated. Proton relaxivity values were obtained in three different homogeneous dispersions of GO-Fe3O4 composites synthesized by precipitating Fe3O4 nanoparticles in three different reaction mixtures containing 0.01 g, 0.1 g, and 0.2 g of graphene oxide. A noticeable difference in proton relaxivity values was observed between the three cases. A comprehensive structural and magnetic characterization revealed discrete differences in the extent of reduction of the graphene oxide and spacing between the graphene oxide sheets in the three composites. The GO-Fe3O4 composite framework that contained graphene oxide with least extent of reduction of the carboxyl groups and largest spacing between the graphene oxide sheets provided the optimum structure for yielding a very high transverse proton relaxivity value. It was found that the GO-Fe3O4 composites possessed good biocompatibility with normal cell lines, whereas they exhibited considerable toxicity towards breast cancer cells. (C) 2015 AIP Publishing LLC.

Automated cell viability assessment using a microfluidics based portable imaging flow analyzer

In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting. (C) 2015 AIP Publishing LLC.

A new approach to depict bone surfaces in finger imaging using photoacoustic tomography

Imaging the vasculature close around the finger joints is of interest in the field of rheumatology. Locally increased vasculature in the synovial membrane of these joints can be a marker for rheumatoid arthritis. In previous work we showed that part of the photoacoustically induced ultrasound from the epidermis reflects on the bone surface within the finger. These reflected signals could be wrongly interpreted as new photoacoustic sources. In this work we show that a conventional ultrasound reconstruction algorithm, that considers the skin as a collection of ultrasound transmitters and the PA tomography probe as the detector array, can be used to delineate bone surfaces of a finger. This can in the future assist in the localization of the joint gaps. This can provide us with a landmark to localize the region of the inflamed synovial membrane. We test the approach on finger mimicking phantoms.

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4 Delta and bud6 Delta alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.

Imaging Flow Cytometry With Femtosecond Laser-Micromachined Glass Microfluidic Channels

Microfluidic/optofluidic microscopy is a versatile modality for imaging and analyzing properties of cells/particles while they are in flow. In this paper, we demonstrate the integration of fused silica microfluidics fabricated using femtosecond laser machining into optofluidic imaging systems. By using glass for the sample stage of our microscope, we have exploited its superior optical quality for imaging and bio-compatibility. By integrating these glass microfluidic devices into a custom-built bright field microscope, we have been able to image red blood cells in flow with high-throughputs and good fidelity. In addition, we also demonstrate imaging as well as detection of fluorescent beads with these microfluidic devices.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

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.