Name: analyzing mixing inhomogeneity in a microfluidic device by microscale schlieren technique
Uploaded: 2015-06-12T14:00:00
Description: Watch this Scientific Journal Video about Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique at JoVE.com

This work was supported by the Ministry of Science and Technology of Taiwan under Grant Number 101-2221-E-002-064-MY3.

1. Fabrication of Microfluidic Device
<ol>
	<li>Use a graphic layout software (e.g., AutoCAD) to draw the outline of a T-microchannel. For the T-microchannel, the two feed channels are 90 &#181;m wide and 2,500 &#181;m long, and the confluence channel is 180 &#181;m wide and 3,000 &#181;m long. Connect the end of each channel to an individual circle with a diameter of 1,100 &#181;m.</li>
	<li>Mark &#8216;clear&#8217; and &#8216;dark&#8217; for the exposure and covered areas, respectively. For a negative photor...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landolt-B&#246;rnstein:+Numerical+Data+and+Functional+Relationships+in+Science+and+Technology.">Landolt-B&#246;rnstein: Numerical Data and Functional Relationships in Science and Technology.</a> Optical Constants: Refractive Indices of Pure Liquids and Binary Liquid Mixtures (Supplement to III/38). 47, (2008).</li><li>Heller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remarks+on+refractive+index+mixture+roles.">Remarks on refractive index mixture roles.</a> Journal of Physical Chemistry. 69 (4), 1123-1129 (1965).</li><li>Korczyk, P. M., Cybulski, O., Makulska, S., Garstecki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unsteadiness+of+the+rates+of+flow+on+the+dynamics+of+formation+of+droplets+in+microfluidic+systems.">Effects of unsteadiness of the rates of flow on the dynamics of formation of droplets in microfluidic systems.</a> Lab on a Chip. 11 (1), 173-175 (2011).</li><li>Dreher, S., Engler, M., Kockmann, N., Woias, P., Bockhorn, H., Mewes, D., Peukert, W., Warnecke, H. .. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+and+experimental+investigations+of+convective+micromixers+and+microreactors+for+chemical+reactions.">Theoretical and experimental investigations of convective micromixers and microreactors for chemical reactions.</a> Micro and Macro Mixing: Analysis, Simulation and Numerical Calculation. , (2010).</li><li>Huang, C. Y., Gregory, J. W., Sullivan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modified+schlieren+technique+for+micro+flow+visualization.">A Modified schlieren technique for micro flow visualization.</a> Measurement Science &amp; Technology. 18 (5), N32-N34 (2007).</li><li>Tyn, M. T., Calus, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+concentration+dependence+of+mutual+diffusion+coefficients+of+some+binary+liquid+systems.">Temperature and concentration dependence of mutual diffusion coefficients of some binary liquid systems.</a> Journal of Chemicaland Engineering Data. 20 (3), 310-316 (1975).</li><li>Celik, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+estimation+and+reporting+of+uncertainty+due+to+discretization+in+CFD+applications.">Procedure for estimation and reporting of uncertainty due to discretization in CFD applications.</a> Journal of Fluids Engineering. 130 (7), 078001 (2008).</li><li>Tasi&#263;, A. &. #. 3. 8. 1. ;., Djordjevi&#263;, B. D., Grozdani&#263;, D. 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For fluidic mixing in a microfluidic device, the microscale schlieren technique is able to measure the magnitude of concentration gradient through quantifying change in light intensity. Because the principle of this technique relies on detecting the alternation of light propagation, the working fluids and the microfluidic device have to be transparent to the incident light. In addition, the protocol requires a linear relationship between the refractive index of the solution and its composition so that preliminary assessm...

Fluid mixing is an important issue that is found in many industrial processes and biological systems. With the emergence of microfluidics, mixing in microscale has brought much attention due to its challenge in diffusion domination among the mass transport mechanisms. Since designing an effective micromixer required quantitative validation, several measuring methods were developed5-7. Nevertheless, the three-dimensional structure, commonly found in efficient micromixers5, demands a more accurate representation of the concentration field that the common measuring techniques fail to deliver. Due to the limit of viewing angle8 or reaction kinetics6, the aforementioned methods may produce misleading results that do not correctly account for the homogeneity of the mixture.
For optically transparent fluids mixing in optically transparent microstructures, microscale schlieren technique3,9-14 provides an attractive alternative to analyze mixing inhomogeneity. In the past, microscale schlieren technique has been mostly used to visualize compressible flow9-13,15 or phase gradient16. Microscale schlieren technique benefits from both a simple optical layout and high sensitivity and enables not only the non-invasive investigation of specific flow feature that causes optical disturbance but is well suited for use in assessing mixing. In this paper, we construct the microscale schlieren system by inserting a knife-edge in the back focal plane of the objective of a microscope, describe a calibration procedure to realize quantitative analysis, and report a validation measurement in a microfluidic oscillator4. To implement measurements, the working fluids are properly selected so that the refractive index of the mixed fluids varies linearly with the composition, and the thickness of the target microfluidic device is identical to the one used in calibration. Besides species concentration, microscale schlieren technique can be extended to measure the gradient of other scalar quantity that is linearly correlated to the index of refraction, such as temperature or salinity.

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			<td>Permanent Epoxy Negative Photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2150</td>
			<td></td>
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		<tr>
			<td>single side polished silicon wafer</td>
			<td>Light Technology</td>
			<td>S4W1PP5SABUP1</td>
			<td>p-type, diameter: 100&#177;0.5 mm, thickness: 525 &#177;25 mm, orientation: (1 0 0)</td>
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			<td>syringe pump</td>
			<td>kdScientific</td>
			<td>kds210</td>
			<td></td>
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			<td>syringe, i.e. 10 ml</td>
			<td>Terumo</td>
			<td>SS-10L2138</td>
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			<td>Hoffman modulation contrast microscope</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED</td>
			<td></td>
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			<td>5X objective lens</td>
			<td>Leica Microsystems</td>
			<td>N PLAN</td>
			<td>NA = 0.12</td>
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			<td>knife-edge</td>
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			<td></td>
			<td>custom made part</td>
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			<td>camera, i.e. high speed</td>
			<td>Integrated Design Tools</td>
			<td>NX7-S1</td>
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			<td>C-mount adapter HC 0.63x</td>
			<td>Leica Microsystems</td>
			<td>541537</td>
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			<td>camera operating software</td>
			<td>Integrated Design Tools</td>
			<td>MotionPro X Studio 2.02.01</td>
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			<td>polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard-184 silicone elastomer kit</td>
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			<td>Teflon tubing, i.e. O.D. x I.D. 1/16 in. x 0.031 in.</td>
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			<td>58700-U</td>
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			<td>micro glass slide</td>
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			<td>hot plate</td>
			<td>Yeong-Shin</td>
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			<td>distilled water, i.e. HPLC grade</td>
			<td>Alps Chemicals</td>
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			<td>ethyl alcohol, i.e. reagent grade</td>
			<td>Nihon Shiyaku Reagent</td>
			<td>EA448652</td>
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			<td>image processing software</td>
			<td>Mathworks</td>
			<td>MATLAB R2009a</td>
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			<td>computational fluid dynamics package</td>
			<td>ESI group</td>
			<td>CFD-ACE+ 2008</td>
			<td></td>
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analyzing mixing inhomogeneity in a microfluidic device by microscale schlieren technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

The grayscale ratio I/I0 under different Reynolds number for both positive and negative gradients of mass fraction is shown (Figure 2) with a symmetrical band appearing in the middle of the T-microchannel. At low Reynolds number, the tail of the schlieren band is expanded and blurred due to the dispersion across the mixing interface. As the Reynolds number increases, the diffusion length shortens leading to a narrower band. At different downstream locations, the variations of...

Watch this Scientific Journal Video about Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique at JoVE.com

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

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We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

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We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

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Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...