This work has been supported by the National S&#38;T Major Project of China (ZX069).

1. Production of MTGS structure with good hermeticity
NOTE: The procedures for MTGS structure include the preparations for components of the combined structure, the heat treatment process, and examinations for the performance of MTGS samples. The complete MTGS structure consists of a steel shell, Kovar conductor, and sealing glass. See the diagram and dimensions shown in Figure 1 and Table 1, respectively.
<ol>
	<li>Pour the granulated glass powder (~1.1 g) into the mold, then place the mold onto the press machine to process the granulated glass as shown in Figure 2a,b.</li>
	<li>Switch on the press machine (push the red button) to compact the granulated glass into the glass cylinder as shown in Figure 2c,d. 
	NOTE: The density control of glass cylinder is important for the performance of the MTGS structure, because too many pores in the glass cylinder will lead to failure of the hermeticity of MTGS structure.</li>
	<li>Place the glass cylinder into the heating furnace to be sintered (see Figure 3).</li>
	<li>The sintered glass cylinder, steel shell, and Kovar conductor are manufactured with a special graphite gasket, as shown in Figure 4. Place this model onto the quartz septum in the heating furnace using a claw for heat treatment (see Figure 4). Keep the cooling rate as 0.5 &#176;C/min to avoid breaking of the optical fiber.</li>
	<li>Use visual inspection to identify the surface topography of sealing glass after retrieving the model from the heating furnace.</li>
	<li>Use the high pressure pipeline to examine the hermeticity of MTGS model. Install the model onto the pipeline by the card sleeve type joint. Slowly change the pressure from 1 MPa to 8 MPa, holding each pressure for 24 h.</li>
	<li>Use the scanning electron microscope (SEM) to identify the microscopic interface between sealing glass and metal parts as shown in Figure 5. Use 15 kV and 500x magnification to observe the interface clearly. 
	NOTE: From the macrography examination and SEM results, the standard maximum heating temperature is set as 450 &#176;C to obtain the MTGS model with good hermeticity. The standard heating treatment is defined as follows: increase the temperature from (room temperature) RT to 450 &#176;C in increments of 5 &#176;C/min, then drop the temperature to RT as 0.5 &#176;C/min.</li>
</ol>
2. Residual stress measurement in sealing glass
NOTE: The FBG sensor is designed as an appropriate method to measure the stress in the MTGS. The grating length of the FBG sensor is 5 mm to match the height of the glass (5 mm) well.
<ol>
	<li>Compact the granulated glass powder into the glass cylinder as described in steps 1.1&#8211;1.2. 
	NOTE: The height of glass cylinder is important, because if the cylinder is too high (&#62;6 mm), it will be difficult to make a through path for the FBG sensor without destroying the glass material.</li>
	<li>Drill the glass cylinder using drill speed of 5,000 rpm to produce three equally spaced through-holes to prepare paths for optical fiber sensors (diameter 0.45 mm). Sinter the glass cylinder with holes using the same heat treatment as shown in Figure 4.</li>
	<li>Manufacture the MTGS model as described in step 1.4. Then, put the fiber through the path in sealing glass and position the grating region of the FBG exactly within the glass. 
	NOTE: Because the flow in the vertical furnace can blow up the grating region, which leads to the mismatching of FBG and glass, the tail of optical fiber must be hung with a small nail to keep the position of FBG accurate.</li>
	<li>Fuse the head of optical fiber with a FC connector by the fusion splicers. Then, match the FC connector with the OPM-T400, which is an interrogator to demodulate the wavelength data and spectrum of FBG. The OPM-T400 is connected to a computer, and the supporting software on the computer can obtain experimental data.</li>
	<li>Process the whole model in a furnace by the standard heat treatment obtained previously. Raise the temperature from RT to 450 &#176;C as 5 &#176;C/min, then drop the temperature to RT in increments of 0.5 &#176;C/min. The grating region will become fused with the sealing glass as it is heated to melt. When the temperature cools down to RT, the glass will solidify and the FBG sensor will become well-fused with the sealing material.</li>
	<li>Record the real-time Bragg wavelength data using the software (shown in Figure 6). The only factor inducing changes of wavelength and spectrum is the residual stress generated in sealing glass, because the temperature before and after this step is both RT.</li>
</ol>
NOTE: The residual stress can be calculated through the strain-wavelength relationship of FBG14 and Hook&#8217;s law, as shown below.
<img alt="Equation 1" src="/files/ftp_upload/60064/60064equ01v2.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/60064/60064equ02v2.jpg" />
Where: the &#916;&#955;B is the Bragg wavelength shift induced by the residual stress, &#955;B is the initial wavelength of FBG, Pe is the strain-optic coefficient, &#949; is the residual strain in the glass, E is the Young&#8217;s modulus of sealing glass, and &#963; is the residual stress in the glass.
3. Preventing the failure of MTGS structure under high temperature
NOTE: When working at a high temperature, the hermeticity of the MTGS structure will be affected, because the thermal expansion of steel shell leads to the decrease of residual stress in sealing glass. Thus, it is possible this protocol can prevent the failure of hermeticity due to the online monitoring of residual stress change in sealing glass.
<ol>
	<li>Manufacture the MTGS model as done in step 1.4. The type of FBG to monitor temperature and stress simultaneously is the fiber Bragg grating array sensor, including two grating regions on one fiber, with a 10 mm distance between these two sensors. 
	NOTE: These two grating are defined as FBG-1 and FBG-2. The initial Bragg wavelengths of FBG-1 and FBG-2 are 1545 nm and 1550 nm, respectively.</li>
	<li>Place FBG-1 into the sintered glass cylinder to monitor the stress and temperature. Place FBG-2 outside the glass to monitor the temperature only, as shown in Figure 7a,b. In this way, FBG-1 is affected by both temperature and residual stress change, and FBG-2 is only affected by temperature of sealing glass.</li>
	<li>Place the MTGS model with optical fiber in the furnace as described in steps 2.2&#8211;2.3. Use the standard heat treatment to process the MTGS model with an embedded FBG sensor.</li>
	<li>Impose temperatures of 100 &#176;C, 200 &#176;C, 300 &#176;C, and 400 &#176;C onto the model and hold each temperature for 100 min.</li>
</ol>
NOTE: FBG-1 monitors the stress and temperature simultaneously expressed as the Bragg wavelength shift &#916;&#955;B-1, and FBG-2 monitors the temperature change by &#916;&#955;B-2 as shown in Figure 8a,b. The relationships between Bragg wavelength shift and measured parameters are shown as follows:
<img alt="Equation 3" src="/files/ftp_upload/60064/60064equ03.jpg" />
<img alt="Equation 4" src="/files/ftp_upload/60064/60064equ04.jpg" />
Where: &#958; is thermo-optic coefficient, &#945; is thermal expansion coefficient of optical fiber, and &#916;T is temperature change before and after the experiment. The &#916;&#955;B-3 induced by residual stress can be separated through subtracting &#916;&#955;B-1 from &#916;&#955;B-2 (see Figure 8c). This is the demodulation method for simultaneous temperature and stress monitoring of sealing glass at high temperatures.
4. Monitoring high pressure
NOTE: The pressure loads on the MTGS structure will have effects on the residual stress in sealing glass, so the MTGS model with the embedded FBG sensor is a potential method to monitor the high pressure change.
<ol>
	<li>Prepare the same MTGS model with the FBG sensor as described in step 2.2&#8211;2.3. After the FBG is well-fused with MTGS model, use the claw to take the model out of the furnace.</li>
	<li>Manufacture the MTGS model with the FBG sensor onto a high pressure helium pipeline by the bite type tube fittings as shown in Figure 9. Adjust the pressure from 1 MPa to 7 MPa by pressure reducing the valve to impose changing pressure loads on the sealing structure.</li>
	<li>The Bragg wavelength shift &#916;&#955;B is recorded as shown in Figure 10. At the same time, the related residual stress change can be calculated using Equation 1 and Equation 2.</li>
</ol>
5. Theoretical analysis of MTGS structure
<ol>
	<li>Use the modeling software to build the 3D model for MTGS structure, and the dimensions are taken from Table 1 to keep the experimental model and theoretical model consistent.</li>
	<li>Import the 3D model into the finite element analysis software. Assign mechanical properties to the steel shell, sealing glass and Kovar conductor, as shown in Table 2.</li>
	<li>The grid type of the whole model is Hex shape (see Figure 11). The mesh method of the sealing glass and steel shell are sweep, and the Kovar conductor is meshed by structured method. Refine the mesh of sealing glass to guarantee the accuracy of theoretical results. The elements number of Kovar conductor, sealing glass and steel shell are 143700, 20350, and 13400, respectively.</li>
	<li>Set the initial increment, minimum increment, and maximum increment of the static analysis step as 0.01, 1.00 x 10-8 and 1.00 x 10-2, respectively.</li>
	<li>Ensure that the interfaces between the sealing glass and metal parts are bounded. First, impose the changing temperature load (from 370 &#176;C to 20 &#176;C) to simulate the solidification progress of the MTGS model. The stress distribution after this process is shown in Figure 12.</li>
	<li>Impose different temperatures (from 100 &#176;C to 400 &#176;C) onto the whole model to simulate the online monitoring experiments under thermal loads. Under the other circumstance, changing pressure loads (from 1 MPa to 7 MPa) are imposed on the sealing glass to simulate the online monitoring under high pressure. The boundary conditions are shown in Figure 13.</li>
	<li>The numerical results of stress and strain distribution of the whole model are obtained from the destination file shown in Figure 14. Extract the analysis path in the sealing glass shown in Figure 13, of which the position is the monitoring path for FBG sensors in Figure 6a to provide comparison with the measuring results by FBG.</li>
</ol>

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The authors have nothing to disclose.

The critical steps for the stress measuring of sealing material of MTGS structure at high temperature and high pressure include 1) manufacturing of the MTGS models with the FBG sensor, of which the grating region is located at the middle of sealing glass; 2) heating of the whole model using a standard heat treatment process, and after the model cools to RT, the FBG sensor will becomes well-fused with MTGS model, and the residual stress can be measured by Bragg wavelength shift; 3) placing of the complete model into the f...

Metal-to-glass sealing is a sophisticated technology that combines interdisciplinary knowledge (i.e., mechanics, materials, and electrical engineering) and is widely applied in aerospace1, nuclear energy2, and biomedical applications3. It has unique advantages such as higher temperature and pressure endurance compared with organic material sealing structures. According to the difference of coefficient of thermal expansion (CTE), MTGS can be divided into two types: matched seal and mismatched seal4. As for the matched seal, the CTE of metal (&#945;metal) and sealing glass (&#945;glass) are nearly the same to reduce the thermal stress in sealing materials. However, to keep good hermeticity and mechanical robustness of the seal structure in harsh environments (i.e., high temperature and high pressure), the mismatched seal displays better performance than the matched seal. Due to the difference between &#945;metal and &#945;glass, the residual stress generates in sealing glass after the annealing process of MTGS structure. If the residual stress is too large (even exceeding the threshold value), the sealing glass displays small defects, such as cracks. If the residual stress is too small, the sealing glass loses its hermeticity. As a result, the value of residual stress is an important measurement.
Analysis of residual stress in MTGS structures has aroused research interests of many groups around the world. The numerical model of axial and radial stress was built based on thin shell theory5. The finite element method was applied to obtain the global stress distribution of an MTGS structure after the annealing process, which was consistent with experimental results6,7. However, because of limitations involving small size and electromagnetic interference, many advanced sensors are not suitable for these circumstances. The indentation crack length method was reported to measure the residual stress in the sealing material of MTG; however, this method was destructive and could not achieve real-time online monitoring of stress changes in glass.
Fiber Bragg grating (FBG) sensors are small in size (~100 &#181;m) and resistant to electromagnetic interference and harsh environments8. In addition, the components of the fiber are similar to those of sealing glass (SiO2), so FBG sensors have no effects on the hermeticity and insulation of the sealing material. FBG sensors have been applied to the residual stress measurement in composite structures9,10,11, and results showed that it displayed good measuring precision and signal response. Simultaneous temperature and stress measurements may be achieved by fiber Bragg grating arrays on one optical fiber12,13.
A novel protocol based on an FBG sensor is demonstrated in this study. The appropriate preparation for the special MTGS structure has been explored by adjusting the maximum heat temperature to ensure the good hermeticity of the MTGS structure. The FBG sensor is embedded in the prepared path of sealing glass to fuse the FBG and glass together after the heat treatment. Then, the residual stress can be obtained by the Bragg wavelength shift of the FBG. The MTGS structure with the FBG sensor is placed under high temperature and high pressure environments to achieve online monitoring of residual stress under changing loads. In this study, the detailed steps to produce an MTS structure with a FBG sensor are outlined. The results show the feasibility of this novel protocol and establish the foundation for the failure diagnosis of an MTGS structure.

<table><tbody><tr><td>ABAQUS</td><td>Dassault SIMULA</td><td>ABAQUS6.14-5</td><td>The software to carry out numerical simulation.</td></tr><tr><td>Fiber Bragg grating sensors</td><td>Femto Fiber Tec</td><td>FFT.FBG.S.00.02 Single</td><td>apodized FBG</td></tr><tr><td>Fusion splicer</td><td>Furukawa Information Technologies and Telecommunications</td><td>S123M12</td><td>FITEL&#039;s line of fusion splicers provides an excellent solution for both field and factory splicing applications?</td></tr><tr><td>Glass powder</td><td>Shenzhen Sialom Advanced Materials Co.,Ltd</td><td>LC-1</td><td>A kind of low melting-point glass powder (380?).</td></tr><tr><td>Graphite mold</td><td>Machining workshop of Tsinghua University</td><td>Graphite</td><td>The mold to locate each part of the metal-to-glass structure.</td></tr><tr><td>Heating furnace</td><td>Tianjin Zhonghuan Electric Furnace Technology Co., Ltd</td><td>SK-G08123-L</td><td>vertical tubular furnace</td></tr><tr><td>Kovar conductor</td><td>Shenzhen Thaistone Technology Co., Ltd</td><td>4J29</td><td>A common material used for the electrical penetration in the metal-to-glass seal structure</td></tr><tr><td>Optical interrogator</td><td>Wuhan Gaussian Optics CO.,LTD</td><td>OPM-T400</td><td>FBG spectrum analysis modules</td></tr><tr><td>Pro/Engineer</td><td>Parametric Technology Corporation</td><td>PROE5.0</td><td>The software to establish the 3D geometry.</td></tr><tr><td>Steel shell</td><td>Beijing Xiongchuan Technology Co., Ltd</td><td>316 stainless steel</td><td>A kind of austenitic stainless steel</td></tr></tbody></table>

optimized sealing process and real-time monitoring of glass-to-metal seal structures

Residual stress is an essential factor to keeping the hermeticity and robustness of a glass-to-metal seal structure. The purpose of this report is to demonstrate a novel protocol to characterize and measure residual stress in a glass-to-metal seal structure without destroying the insulation and hermeticity of sealing materials. In this research, a femto-laser inscribed fiber Bragg grating sensor is used. The glass-to-metal seal structure that is measured consists of a metal shell, sealing glass, and Kovar conductor. To make the measurements worthwhile, the specific heat treatment of metal-to-glass seal (MTGS) structure is explored to obtain the model with best hermeticity. Then, the FBG sensor is embedded into the path of sealing glass and becomes well-fused with the glass as the temperature cools to RT. The Bragg wavelength of FBG shifts with the residual stress generated in sealing the glass. To calculate the residual stress, the relationship between Bragg wavelength shift and strain is applied, and the finite element method is also used to make the results reliable. The online monitoring experiments of residual stress in sealing glass are carried out at different loads, such as high temperature and high pressure, to broaden functions of this protocol in harsh environments.

From the results of Figure 5, the standard heat treatment to produce the MTGS models with high pressure endurance is explored, and the models can satisfy the examinations (i.e., light transmissions, pressure endurance, SEM, etc.). Thus, the produced MTGS structure can be applied to keep hermeticity in harsh environments.
The FBG can be well-fused with MTGS structure, and the residual strain in sealing glass will be reflected by Bragg wavelength shift after the hea...

Watch this Scientific Journal Video about Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures at JoVE.com

Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures

Key procedures to optimize the sealing process and achieve real-time monitoring of the metal-to-glass seal (MTGS) structure are described in detail. The embedded fiber Bragg grating (FBG) sensor is designed to achieve online monitoring of temperature and high-level residual stress in the MTGS with simultaneous environmental pressure monitoring.

Residual stress is an essential factor to keeping the hermeticity and robustness of a glass-to-metal seal structure. The purpose of this report is to ...

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7.3K Views.  Tsinghua University. Dit protocol kan worden gebruikt om de real-time gelijktijdige temperatuur- en stressbewaking van de metaal-glasstructuren voor de eerste keer uit te voeren. Het belangrijkste voordeel van deze techniek is dat de vezel Bragg roostersensor goed kan worden versmolten met de metaal-glas structuur zonder de isolatie of hermeticiteit van de metaal-naar-glas structuur te vernietigen. Het demonstreren van de procedure met Zhichun Fan zal Kangjia Hu, een master student van INET.Om gegranuleerd...

Video: Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengineering

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Biology

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Chemistry

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs

Pressure limitations of many microfluidic platforms have been a significant challenge in microfluidic experimental studies of fractured media. As a result, these platforms have not been fully exploited for direct observation of high-pressure transport in fractures. This work introduces microfluidic platforms that enable direct observation of multiphase flow in devices featuring surrogate permeable media and fractured systems. Such platforms provide a pathway to address important and timely questions such as those related to CO2 capture, utilization and storage. This work provides a detailed description of the fabrication techniques and an experimental setup that may serve to analyze the behavior of supercritical CO2 (scCO2) foam, its structure and stability. Such studies provide important insights regarding enhanced oil recovery processes and the role of hydraulic fractures in resource recovery from unconventional reservoirs. This work presents a comparative study of microfluidic devices developed using two different techniques: photolithography/wet-etching/thermal-bonding versus Selective Laser-induced Etching. Both techniques result in devices that are chemically and physically resistant and tolerant of high pressure and temperature conditions that correspond to subsurface systems of interest. Both techniques provide pathways to high-precision etched microchannels and capable lab-on-chip devices. Photolithography/wet-etching, however, enables fabrication of complex channel networks with complex geometries, which would be a challenging task for laser etching techniques. This work summarizes a step-by-step photolithography, wet-etching and glass thermal-bonding protocol and, presents representative observations of foam transport with relevance to oil recovery from unconventional tight and shale formations. Finally, this work describes the use of a high-resolution monochromatic sensor to observe scCO2 foam behavior where the entirety of the permeable medium is observed simultaneously while preserving the resolution needed to resolve features as small as 10 &#181;m.

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs

This paper describes a protocol along with a comparative study of two microfluidic fabrication techniques, namely photolithography/wet-etching/thermal-bonding and Selective Laser-induced Etching (SLE), that are suitable for high-pressure conditions. These techniques constitute enabling platforms for direct observation of fluid flow in surrogate permeable media and fractured systems under reservoir conditions.

Pressure limitations of many microfluidic platforms have been a significant challenge in microfluidic experimental studies of fractured media. As a ...

Determining the Mechanical Strength of Ultra-Fine-Grained Metals

The mechanical strengthening of metals is the long-standing challenge and popular topic of materials science in industries and academia. The size dependence of the strength of the nanometals has been attracting a lot of interest. However, characterizing the strength of materials at the lower nanometer scale has been a big challenge because the traditional techniques become no longer effective and reliable, such as nano-indentation, micropillar compression, tensile, etc. The current protocol employs radial diamond-anvil cell (rDAC) X-ray diffraction (XRD) techniques to track differential stress changes and determine the strength of ultrafine metals. It is found that ultrafine nickel particles have more significant yield strength than coarser particles, and the size strengthening of nickel continues down to 3 nm. This vital finding immensely depends on effective and reliable characterizing techniques. The rDAC XRD method is expected to play a significant role in studying and exploring nanomaterial mechanics.

The protocol presented here describes the high-pressure radial diamond-anvil-cell experiments and analyzing the related data, which are essential for obtaining the mechanical strength of the nanomaterials with a significant breakthrough to the traditional approach.

The mechanical strengthening of metals is the long-standing challenge and popular topic of materials science in industries and academia. The size ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Optimized Sealing Process and Real-Time Monitoring of Glass-to-Metal Seal Structures

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