Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Demonstration of a microfluidic polarimeter

Traditional methods of detecting chiral molecules, such as optical rotation are not suitable for miniaturization, since, the magnitude of the rotation of polarization scales down linearly with the optical path length of the device. Since the origin of optical activity is due to difference of refractive indices between the two circularly polarized states of light, it is possible to detect chiral media by measuring the dependence of the angles of refraction on the polarization state of the incident light. This however is a weak effect and hence requires sensitive optical detection schemes, based on novel polarization modulation techniques. The device can be scaled down for applications involving small sample volumes. Fabrication details of a prototype microfluidic device are described.

Charge Injection through Nanocomposite Electrode in Microfluidic Channel for Electrical Lysis of Biological Cells

Several concepts have been developed in the recent years for nanomaterial based integrated MEMS platform in order to accelerate the process of biological sample preparation followed by selective screening and identification of target molecules. In this context, there exist several challenges which need to be addressed in the process of electrical lysis of biological cells. These are due to (i) low resource settings while achieving maximal lysis (ii) high throughput of target molecules to be detected (iii) automated extraction and purification of relevant molecules such as DNA and protein from extremely small volume of sample (iv) requirement of fast, accurate and yet scalable methods (v) multifunctionality toward process monitoring and (vi) downward compatibility with already existing diagnostic protocols. This paper reports on the optimization of electrical lysis process based on various different nanocomposite coated electrodes placed in a microfluidic channel. The nanocomposites are synthesized using different nanomaterials like Zinc nanorod dispersion in polymer. The efficiency of electrical lysis with various different electrode coatings has been experimentally verified in terms of DNA concentration, amplification and protein yield. The influence of the coating thickness on the injection current densities has been analyzed. We further correlate experimentally the current density vs. voltage relationship with the extent of bacterial cell lysis. A coupled multiphysics based simulation model is used to predict the cell trajectories and lysis efficiencies under various electrode boundary conditions as estimated from experimental results. Detailed in-situ fluorescence imaging and spectroscopy studies are performed to validate various hypotheses.

MECHANISM OF CELL LYSIS IN MICROFLUIDIC CHANNEL WITH INTEGRATED NANOCOMPOSITE ELECTRODES

This paper reports on the characterization of an integrated micro-fluidic platform for controlled electrical lysis of biological cells and subsequent extraction of intracellular biomolecules. The proposed methodology is capable of high throughput electrical cell lysis facilitated by nano-composite coated electrodes. The nano-composites are synthesized using Carbon Nanotube and ZnO nanorod dispersion in polymer. Bacterial cells are used to demonstrate the lysis performance of these nanocomposite electrodes. Investigation of electrical lysis in the microchannel is carried out under different parameters, one with continuous DC application and the other under DC biased AC electric field. Lysis in DC field is dependent on optimal field strength and governed by the cell type. By introducing the AC electrical field, the electrokinetics is controlled to prevent cell clogging in the micro-channel and ensure uniform cell dispersion and lysis. Lysis mechanism is analyzed with time-resolved fluorescence imaging which reveal the time scale of electrical lysis and explain the dynamic behavior of GFP-expressing E. coli cells under the electric field induced by nanocomposite electrodes. The DNA and protein samples extracted after lysis are compared with those obtained from a conventional chemical lysis method by using a UV-Visible spectroscopy and fluorimetry. The paper also focuses on the mechanistic understanding of the nano-composite coating material and the film thickness on the leakage charge densities which lead to differential lysis efficiency.

MRT Letter: Light Sheet Based Imaging Flow Cytometry on a Microfluidic Platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state-of-art techniques the proposed technique shows marked improvement. Using fluorescently-coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on-the-go. Overall, the proposed system is cost-effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101-1107, 2013. (c) 2013 Wiley Periodicals, Inc.

Suppression of air bubble in microfluidic channel using electro-acoustic excitation

A simple method to study the air bubble dynamics and to burst the air bubbles formed on the electrode– electrolyte interface in a parallel gate electrode fluidic channel is demonstrated. Upon application of a voltage across the electrodes,volume of water contained between them begins to electrolyzing depending on the conductivity, as well as it boils due to heating effect. This results in bubble formation within. These bubbles grow in radius with higher potential difference applied across the electrodes. As an approach towards removing these bubbles, an alternating current is applied at low potential difference of a 5 volts and high frequency at few megahertz. The alternating electric field had a heating effect on the bubbles where the energy input due to current heats up water and bursts the bubble. The bubbles of size up to 480μm were burst at 2500 V/m using this approach.

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.

Single-Cell Optical Absorbance Characterization With High-Throughput Microfluidic Microscopy

Morphological changes in cells associated with disease states are often assessed using clinical microscopy. However, the changes in chemical composition of cells can also be used to detect disease conditions. Optical absorption measurements carried out on single cells using inexpensive sources, detectors can help assess the chemical composition of cells; thereby enable detection of diseases. In this article, we present a novel technique capable of simultaneously detecting changes in morphology and chemical composition of cells. The presented technique enables characterization of optical absorbance-based methods against microscopy for detection of disease states. Using the technique, we have been able to achieve a throughput of about 1000 cells per second. We demonstrate the proof-of-principle by detecting malaria in a given blood sample. The presented technique is capable of detecting very lower levels of parasitemia within time scales comparable to antigen-based rapid diagnostic tests.

High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry

In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per mu l) obtained from our instrument, with that of a commercially available hematology analyzer.

Fabrication of Microfluidic-Nanofluidic Channels with Integrated Electrodes

We report on the fabrication of microfluidc-nanofluidic channels on Si incorporated with embedded metallic interconnects. The device aids the study of motion of dispersed particles relative to the fluid under the influence of spatially uniform electric field. Optical lithography in combination with focused ion beam technique was used to fabricate the microfluidic-nanofluidic channels, respectively. Focused ion beam technique was also used for embedding the electrodes in the nanochannel. Gold contact pads were deposited using sputtering. The substrate was finally anodically bonded to a glass substrate.

Analysis of integrated optofluidic lab-on-a-chip sensor based on refractive index and absorbance sensing

The analysis of a fully integrated optofluidic lab-on-a-chip sensor is presented in this paper. This device is comprised of collinear input and output waveguides that are separated by a microfluidic channel. When light is passed through the analyte contained in the fluidic gap, optical power loss occurs owing to absorption of light. Apart from absorption, a mode-mismatch between the input and output waveguides occurs when the light propagates through the fluidic gap. The degree of mode-mismatch and quantum of optical power loss due to absorption of light by the fluid form the basis of our analysis. This sensor can detect changes in refractive index and changes in concentration of species contained in the analyte. The sensitivity to detect minute changes depends on many parameters. The parameters that influence the sensitivity of the sensor are mode spot size, refractive index of the fluid, molar concentration of the species contained in the analyte, width of the fluidic gap, and waveguide geometry. By correlating various parameters, an optimal fluidic gap distance corresponding to a particular mode spot size that achieves the best sensitivity is determined both for refractive index and absorbance-based sensing.

Synthesis of pure iron magnetic nanoparticles in large quantity

Free nanoparticles of iron (Fe) and their colloids with high saturation magnetization are in demand for medical and microfluidic applications. However, the oxide layer that forms during processing has made such synthesis a formidable challenge. Lowering the synthesis temperature decreases rate of oxidation and hence provides a new way of producing pure metallic nanoparticles prone to oxidation in bulk amount (large quantity). In this paper we have proposed a methodology that is designed with the knowledge of thermodynamic imperatives of oxidation to obtain almost oxygen-free iron nanoparticles, with or without any organic capping by controlled milling at low temperatures in a specially designed high-energy ball mill with the possibility of bulk production. The particles can be ultrasonicated to produce colloids and can be bio-capped to produce transparent solution. The magnetic properties of these nanoparticles confirm their superiority for possible biomedical and other applications.

Detection of Target DNA using Photo-Reactive Protoporphyrin Moeity on a Nanocomposite Substrate

Detection of pathogens from infected biological samples through conventional process involves cell lysis and purification. The main objective of this work is to minimize the time and sample loss, as well as to increase the efficiency of detection of biomolecules. Electrical lysis of medical sample is performed in a closed microfluidic channel in a single integrated platform where the downstream analysis of the sample is possible. The device functions involve, in a sequence, flow of lysate from lysis chamber passed through a thermal denaturation counter where dsDNA is denatured to ssDNA, which is controlled by heater unit. A functionalized binding chamber of ssDNA is prepared by using ZnO nanorods as the matrix and functionalized with bifunctional carboxylic acid, 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) which is further attached to a linker molecule 1-ethyl-3-(3-dimethylaminopropyl) (EDC). Linker moeity is then covalently bound to photoreactive protoporphyrin (PPP) molecule. The photolabile molecule protoporphyrin interacts with -NH2 labeled single stranded DNA (ssDNA) which thus acts as a probe to detect complimentary ssDNA from target organisms. Thereafter the bound DNA with protoporphyrin is exposed to an LED of particular wavelength for a definite period of time and DNA was eluted and analyzed. UV/Vis spectroscopic analysis at 260/280 nm wavelength confirms the purity and peak at 260 nm is reconfirmed for the elution of target DNA. Quantitative and qualitative data obtained from the current experiments show highly selective detection of biomolecule such as DNA which have large number of future applications in Point-of-Care devices.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro-to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine. (C) 2014 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.