ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

MICROFLUIDICS LAB ON CHIP

ABSTRACT
Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time series data for the governing regulatory proteins in a large number of individual living cells. An ideal data acquisition system would allow for the growth of a large population of cells in a dened environment which can be monitored by high resolution microscopy for an extended period of time. Thus this lab will consist on a brief theory about microfluidics then will follow the practical work going from the chip fabrication to one of its applications: the tracking or monitoring of particles (beads or E. coli) in this device and the subsequent analysis of the acquired data. Some imaging techniques will also be introduced. Finally, a few questions will be discussed in order to outline some important points.

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

TABLE OF CONTENTS
1 Theory............................................................................................................................................. 3 1.1 1.2 1.3 1.4 2 Basic Principles of Microfluidics ............................................................................................ 4 Device Fabrication .................................................................................................................. 5 Questions (Theory) .................................................................................................................. 8 Integration of Microuidics and Microscopy .......................................................................... 9

Practical work .............................................................................................................................. 11 2.1 2.2 2.3 2.4 2.5 2.6 Material requirements ............................................................................................................ 11 PDMS silicon mold ............................................................................................................... 11 Integration of Microuidics and Microscopy ........................................................................ 14 Run the sample ...................................................................................................................... 16 Viewing / Tracking Particles in Device Geometry ................................................................ 17 Epifluorescence ..................................................................................................................... 18

Data analysis ................................................................................................................................ 19 3.1 3.2 3.3 3.4 3.5 Calibration ............................................................................................................................. 19 Particle Tracking ................................................................................................................... 19 Matlab analysis ...................................................................................................................... 23 Particle analysis ..................................................................................................................... 23 Questions ............................................................................................................................... 26

References .................................................................................................................................... 26

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

1. THEORY
Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time series data for the governing regulatory proteins in a large number of individual living cells. An ideal data acquisition system would allow for the growth of a large population of cells in a dened environment which can be monitored by high resolution microscopy for an extended period of time. In this laboratory exercise we fabricate and use such a data acquisition system. With our setup, the gene expression state of each cell could be monitored for the length of the experiment, giving the experimenter accurate data about the temporal progression of each individual cell within the larger population. To this end, bioengineers have increasingly used devices with uid channels on the micron scale known as microuidic devices. The goal of this exercise is to fabricate and use such a microuidic device.

Figure 1. Microfluidics provide a tool for the miniaturization and serial processing of fluids allowing better control of their properties, integration of different operations and parallelization. Reproduced from 1

Microtechnology in general, and microuidics in particular, can facilitate the accurate study of cellular behavior in vitro because it provides the necessary tools for recreating in vivo-like cellular microenvironments. Microuidics involve the handling and manipulation of very small uid volumes, enabling creation and control of microliter-volume reactors while drawing advantages from low thermal mass, efficient mass transport, and large surface area-to-volume ratios. Because uid viscosity, not inertia, dominates uid behavior at this scale, microuidic ow is laminar, ensuring that the system does not include turbulent ows which would be detrimental for observing cellular behavior under high magnication. Lately, microuidic lab-on-a-chip devices have become increasingly valuable as the known complexity of gene networks grows, driving the need for reduced-scale assays in probing entire parameter spaces of genetic circuits. The result has been the development of integrated microuidic circuits analogous to their electrical counterparts, which aim to support large-scale multi-parameter analysis in parallel. Recent applications of microuidics in biotechnology include DNA amplication, purication, separation 2, and sequencing3; large-scale proteomic analysis4; development of memory storage devices5; cell sorting6 and single-cell gene expression proling. The use of microfluidic devices to conduct biomedical research and create clinically useful technologies has a number of significant advantages. First, because the volume of fluids within these channels is very small, usually several nanoliters, the amount of reagents and analytes used is quite small. This is especially significant for expensive reagents. The fabrication techniques used to construct microfluidic devices (discussed in more depth later) are relatively inexpensive and very amenable both to: highly elaborate multiplexed devices and mass

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

production. In a manner similar to that for microelectronics, microfluidic technologies enable the fabrication of highly integrated devices for performing several different functions on the same substrate chip. One of the long term goals in the field of microfluidics is to create integrated, portable clinical diagnostic devices for home and bedside use, thereby eliminating time consuming laboratory analysis procedures.

1.1 Basic Principles of Microfluidics 1.1.1 Reynolds number

The flow of a fluid through a microfluidic channel can be characterized by the Reynolds number, defined as equation (1.1)

Re

L Vavg

(1.1)

Where L is the most relevant length scale, is the viscosity, is the fluid density, and Vavg is the average velocity of the flow. For many microchannels, L is equal to 4A/P where A is the cross sectional area of the channel and P is the wetted perimeter of the channel. Due to the small dimensions of microchannels, the Re is usually much less than 100, often less than 1. In this low Reynolds number regime, flow is completely laminar and no turbulence occurs the transition to turbulent flow generally occurs in the range of Reynolds number 2000. Laminar flow provides a means by which molecules can be transported in a relatively predictable manner through microchannels. Note, however, that even at Reynolds numbers below 100, it is possible to have momentum-based phenomena such as flow separation.

1.1.2 Poiseuilles Law

In such a laminar flow of viscous and incompressible fluid, the pressure drop and the flow rate, as well as the effective resistance might be obtained by using the Poiseuille equation (1.2).

p=

8LQ r 4

and

R=

8x r 4

(1.2)

Where p is the pressure drop, Q is the volumic flow rate, R is the resistance to flow, L is the length of the channel, r radius of the channel, is the dynamic fluid viscosity and x the distance in direction of flow.

1.1.3 Pressure Driven Flow

There are two common methods by which fluid actuation through microchannels can be achieved. In pressure driven flow, the fluid is pumped through the device via positive displacement pumps, such as syringe pumps. One of the basic laws of fluid mechanics for pressure driven laminar flow, the so-called no-slip boundary condition, states that the fluid velocity at the walls must be zero. This produces a parabolic velocity profile within the channel (Figure 2.a). The parabolic velocity profile has significant implications for the distribution of molecules transported within a channel. Pressure driven flow can be a relatively inexpensive and quite reproducible approach to pumping fluids through microdevices. With the increasing efforts at developing functional micropumps, pressure driven flow is also amenable to miniaturization. (Figure 2.a)

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

1.1.4 Electrokinetic Flow

Another common technique for pumping fluids is that of electroosmotic pumping. If the walls of a microchannel have an electric charge, as most surfaces do, an electric double layer of counter ions will form at the walls. When an electric field is applied across the channel, the ions in the double layer move towards the electrode of opposite polarity. This creates motion of the fluid near the walls and transfers via viscous forces into convective motion of the bulk fluid. If the channel is open at the electrodes, as is most often the case, the velocity profile is uniform across the entire width of the channel (Figure 2.b). However, if the electric field is applied across a closed channel (or a backpressure exists that just counters that produced by the pump), a recirculation pattern forms in which fluid along the center of the channel moves in a direction opposite to that at the walls (Figure 2.c). In closed channels, the velocity along the centerline of the channel is 50% of the velocity at the walls.
a b

Figure 2 a) Velocity profile in a microchannel with aspect ratio 2:5 under conditions of pressure driven flow. Note that the velocity is assumed to be zero at the walls in most treatments of transport of liquids. b) The very uninteresting flow velocity profile calculated for electroosmotic pumping in an open channel. Such a channel (in the absence of backpressure) exhibits plug flow. Shown in the situation for negatively charged walls; the anode is at the left and the cathode is at the right. In fact the profile is very interesting close to the walls, since velocity drops to zero at the walls over a distance that is comparable to the thickness of the electrical double layer. c) The view of the electroosmotic flow velocity vectors in a closed channel. Note that the recirculation results in equal total flows to the right and left at all vertical planes through the channel. The anode is on the left and the cathode is on the right and the walls are negatively charged.

1.2 Device Fabrication 1.2.1 Photolithography

In recent years, soft lithography has become the preferred method for fabricating microuidic devices for biology. Soft lithography includes a suite of methods for replicating a pattern using elastomeric polymers (Figure 4). Soft lithography can be represented as a three-step process comprised of concept developing, rapid prototyping, and replica molding. The rst step, concept developing, involves drafting a device design in a computer-aided design (CAD) program. Here, a general idea for a device that serves some purpose is eshed out using engineering approaches. Using the laws of uid dynamics under the condition of low Reynolds number for microvolume ow, uid channel resistances are calculated and modied to satisfy desired driving pressures and ow rates. Following ne-tuning of the entire channel architecture, the device design is broken up into multiple layers, where all features of a given height are placed on a single layer for photolithographic purposes. Finally, all the device layers are printed at high resolution onto transparency lm. These are then fastened to ultra-transmissive borosilicate glass for use as a photomask set in the following contact lithography step. Or alternatively, as we did in the CMI (Center of

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

MicroNanoTechnology EPFL) for this laboratory, a photomask may be created by patterning chrome on a glass plate. For the design we have chosen the recently proposed microfluidics chip that has been used for monitoring the collective synchronization properties in an engineered gene network with global intercellular coupling in a growing population of cells that exhibit spatiotemporal waves occurring at millimeter scales 7. The chip design is shown in Figure 3.

Figure 3. Microfluidic device used for maintaining E. coli or beads at a constant density. The main channel (blue) supplies media to cells in the trapping chamber, and the flow rate can be externally controlled to change the effective rates of an engineered gene network7.

There is presented the lithography concept to understand the how have been made the wafer that you will use to fabricate your device. Due to the time constraints of this exercise, this part of fabrication is already made by TA. In rapid prototyping, a positive or negative photoresist is spin coated onto a clean silicon wafer at a specied thickness and then exposed to UV light through the photomask to selectively crosslink the features represented by the mask. Since each exposure iteration creates all device features of a given height (being the depth of the photoresist layer); this process can be repeated to pattern the wafer for multi-layer device features. The nal result is a positive relief of photoresist on the silicon wafer, known as a master mold, whose topology precisely reects the desired device channel and feature structures and can be used repeatedly to form successive batches of devices. Fabrication of this master mold completes the rapid prototyping step of soft lithography. The nal step, called replica molding, involves the casting of a transparent, silicone-based liquid prepolymer (usually PDMS) against the master mold to generate a negative replica of the master. The prepolymer is rst poured onto the wafer and heat-cured in place to form a rubbery silicone solid. This silicone monolith is then peeled from the mold to reveal the inverted feature topology represented by the mold. For example, ridges on the master mold appear as valleys in the replica. This monolith is then diced into individual devices, bored with a cylindrical punch to form holes for connection to uid reservoirs, and cleaned using Scotch tape and methanol. In the nal step, the feature sides of the devices, along with opposing coverslip surfaces, are briey treated with low power oxygen plasma. This process activates the surfaces of the PDMS devices and glass coverslips so that they form a permanent bond when placed in contact. In bonding the two objects, uid channels in the PDMS are sealed against the at coverslip surface to form microchannels internally connecting the device uidic ports. These nished devices mark completion of the replica molding step of soft lithography

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

or create chrome mask

Concept copied form Danino et al. Nature 463, 326-330 (2010)

Your TAs prepared wafers beforehand

Your will fabricate microfluidic device from this point !

Figure 4. Schematic of microuidic device fabrication using soft lithography (adapted from Ref. 8)

1.2.2 Multilayer soft lithography

The techniques described here can be extended to perform multilayer soft lithography, which provides the capability to bond multiple patterned layers of elastomer to create active microuidic systems containing on-off valves, switching valves, and pumps. There will not be any such sophisticated components in our device but, it is always good to know for your future research since it is more and more used in laboratories. In multilayer soft lithography, in addition to a layer of microchannels cast in PDMS as described before, a second, deformable thin membrane of PDMS is cast by spin coating PDMS onto a master mold. This allows for a layer with a thickness of only about 50 to 100 microns. The thin PDMS layer is then partially cured, and bonded to the thick PDMS layer. Both layers are then bonded to a flat substrate.

Figure 5. a) Multilayer soft lithography fabrication process. A microchannel layer is molded in a thin deformable PDMS membrane through a spin-coating process. A second layer of microchannels is molded from a thick layer of PDMS. The two PDMS layers are bonded together, and the structure is then bonded to a flat substrate. b) Example of a peristaltic pump fabricated from multilayer soft lithography. By successive pressurization of the upper control layer channels, fluid is pumped through the lower fluidic layer. (Adapted from Ref. 1)

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

The end result is a set of microchannel layers separated vertically by a thin membrane of PDMS. The advantage of this architecture is that air or fluid pressure in one of the microchannels can be used to deform the membrane, blocking or constricting fluid flow in the second microchannel. This allows for simple integration of valves and pumps into these multilayered fluidic structures. An overview of the fabrication process and an example of a peristaltic pump are illustrated in Fig. 5. Recent research in the microuidics eld has produced several examples of complex devices with hugely parallel active channel structures for high-throughput cell analysis. In approaching years, the fundamental benets of soft lithography for biology, which include ease of fabrication, inexpensive production, and rapid device turnover, will continue to aid the researcher seeking increasingly functional cell assays.

1.3 Questions (Theory)

Q1. How does the laminar flow help microfluidic design? Why?

Q2. Which network has equal flow through branches? Why? How is it designed in your chip?

Q3. Which path will have higher flow? Why?

Q4. For what are the hooks between media input and waste outputs useful?

Q5. Why do we have 2 inlets and 2 outlets?

Q6. Define low Reynolds number. Typical E.coli (2.0m long and 0.5m in diameter) is characterized by low or high Reynolds number?

Q7. How are fluidic resistance and channel width related?

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

1.4 Integration of Microuidics and Microscopy

Microuidics has recently found wide applications in research aimed at observing cellular development within dynamic microenvironments. Devices designed for these purposes frequently possess the ability to generate thermal and/or chemical gradients across the cell development volume. Another recently demonstrated strength of microuidics is the ability to generate large-scale and highly parallel integrated circuits of fluidic channels for high-throughput cellular analysis11-13. However, for researchers interested in studying the behavior of synthetic gene circuits, the most challenging goal of microuidics has been in supporting long-term single-cell analysis for large sample populations. Therefore, much recent research has focused on this goal using various design strategies. One group approached the difficulties in single-cell analysis by developing a microuidic network enabling the passive and gentle separation of a single cell from bulk suspension 14. This individual cell is focused by hydrostatic pressure and laminar flow streams to a trapping region, where integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Whereas this research focused on individual cells over a relatively short time span, another group developed a microuidic platform for long-term cell culture studies spanning the entire differentiation process of mammalian cells 15. They demonstrated operation of this device by observing a culture of muscle cells differentiating from myoblasts to myotubes over the course of two weeks. To researchers interested in long-term gene expression variability within single-celled prokaryotic and eukaryotic populations, a chemostat likely represents the ideal cell assay. In recent years, the many challenges involved in operating continuous macroscale bioreactors (such as the need for large quantities of reagents) have driven the miniaturization of these devices into microuidic chip-based formats. In continually providing fresh nutrients and removing cellular waste to support exponential growth, the microuidic chemostat (small cell trapping region) presents a nearly constant environment that is ideal for long-term cell culture monitoring with single-cell resolution. Recently, one group presented a microuidic chemostat for culturing bacterial and yeast cells in an array of shallow microscopic chambers with support for dynamically-dened media16. Similarly, a recent implementation of a microuidic bioreactor has enabled long-term culturing and monitoring of small populations of bacteria with single-cell resolution17. This microchemostat contained an integrated peristaltic pump and a series of micromechanical valves to add medium, remove waste, and recover cells. The device was used to observe the dynamics of an E. coli strain carrying a synthetic population control circuit that regulates cell density through a feedback mechanism based on quorum sensing. A nal implementation of the chemostat design was utilized to precisely control and constrain exponential growth of the yeast S.cerevisiae and E. coli to a monolayer18. Here, dimensions of the chemostat device were precisely controlled to constrain exponential growth of yeast and E. coli cells to a monolayer. The device has been modified for imaging a culture of cells growing in exponential phase for many generations. The construction was such that a shallow trapping region will constrain a population of cells to the same focal plane. The significant advantage of monolayer growth in a height-constrained chamber was demonstrated by visualization of a group of cells residing at the trapping region boundary. Through directed planar growth, the researchers were able to resolve the temporal evolution of single-cell gene expression levels with the aid of segmentation and tracking software. Advantages of this device design and software package included simple operation and automated single-cell fluorescence trajectory extraction. Such novel data should prove useful in investigating the timing and variability of gene expression within various synthetic gene regulatory network architectures on the time scale of many cellular generations.

ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

1.4.1 Fluorescence imaging

Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity from nonfluorescing material. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer. Different molecules can now be stained with different colors, allowing multiple types of molecule to be tracked simultaneously. These factors combine give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging. Fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence instead of, or in addition to, reflection and absorption. In most cases, a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as GFP, Green Fluorescent Protein). GFP is a fluorescent protein that was first found in the jellyfish Aequorea Victoria. It has the useful property that its formation is not species specific. This means that it can be fused to virtually any target protein by genetically encoding its cDNA as a fusion with the cDNA of the target protein. This can be done in a live cell, and hence the movement of individual cellular components can now be analyzed across time.
a)

b)

Wavelenght (nm)

Spectrum

Figure 6. a) Fluorescence imaging principle (Wikipedia: Fluorescence_microscopy) b) Excitation and emission spectra of the dyes used in this practical FITC very close the GFP excitation and emission spectra

There is no requirement to fix and permeablize the cells first. The discovery of GFP has made the imaging of real-time dynamic processes commonplace, and caused a revolution in optical imaging. The GFP revolution goes even further with the development of different colored GFP isoforms, such as yellow GFP and cyan GFP. This allows multiple proteins to be viewed simultaneously in a cell. In this practical we use 2.5 m PeakFlow green flow cytometry reference beads that stained with fluorescent dye (FITC) that have been carefully selected to produce emission peaks coincident with labeled cells used in typical flow cytometry applications (GFP labeled cells). Because PeakFlow beads are highly uniform with respect to both size and fluorescence intensity, and because they approximate the size, emission wavelength and intensity of many biological samples, they can be used to calibrate a flow cytometers laser source, optics, stream flow and cell sorting system without wasting valuable and sensitive experimental material. The specimen is illuminated with light of a specific wavelength which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a dichroic mirror. Typical components of a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp), the excitation filter, the

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

dichroic mirror (or dichromatic beamsplitter), and the emission filter. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.

1.4.2Filters
Filters used in this work are called FITC (Figure 7) according to the traditional fluorochromes that were earlier commonly used for green and red fluorescence. In the figure, the blue (1) curve shows the excitation i.e. the wavelengths that illuminate the sample. The red (2) curve shows the emission i.e. the wavelengths that are shown to the viewer.

Figure 7. FITC filter spectrum. 1 = excitation band, 2 = emission band

2. PRACTICAL WORK
2.1 Material requirements
Handling. Safety glasses, gloves, tweezers, Petri dishes, pipettes, spoons, cups, razor blades, scalpels, aluminum foil. Machines. Ventilated fume hood, high precision scale, nitrogen gun, mechanical mixer, vacuum desiccators, manual hole-punching machine, binocular, oven/hot plate, oxygen plasma. Products. Sylgard 184 silicone base, Sylgard curing agent, silanizing agent (TMCS: Chlorotrimethylsilane, 33014 from sigma)

2.2 PDMS silicon molds

The first step in PDMS molding is designing molds and creating them. SU-8 processing and silicon etching are the two protocols commonly used to realize molds for PDMS micro-molding. Procedures for creating these molds are not presented in this present document. Our TAs prepared molds and they are in the AMBL marked wafer holder.

2.2.1

Surface conditioning

The surface conditioning of the mold is important to prevent PDMS sticking. A silanization allows passivation of the surfaces to aid release from PDMS.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

TMCS is corrosive - causes skin burns, harmful in contact with skin also it is armful if swallowed and it is respiratory irritant. Therefore its handling should be done under the fume hood and you are suggested to wear extra pair of gloves. 1) Put on single use additional gloves and operate only under the fume hood. 2) Place a few drops of TMCS in the small glass receptacle located in the desiccator (single use pipettes are available for that purpose).

Wafer with PDMS mold

single use pipettes

TMCS

Note: If TMCS bottle is not in the glass desiccator, fetch it in the solvent cabinet located on the right side of the wet bench.
Desiccator

Glass receptacle

3) 4) 5)

6) 7)

8)

Remove any dust on the surface of the mold using a nitrogen gun. Place the silicon/SU8 mold in this very same desiccator. Close the desiccator and place it under vacuum (this causes the TMCS to evaporate and to form a passivation layer on the mold surface). Close well the TMCS bottle (use tape also). Fill-in the chemicals follow-up document. When desired time is reached (~15min), vent the desiccator. DO NOT breath directly above the open desiccator. Take your mold back, put the TMCS bottle in the desiccator and put it back under vacuum.

15 min

2.2.2 Mixing Degassing

Silicone prepolymer material is very viscous and sticky. Use additional gloves before handling the liquid PDMS. Aluminum foil is used as a liner for protecting equipment in contact with the (Petri dishes, scale, etc.). A single use plastic cup is to be used for preparing the PDMS mixture. The plastic cups are compatible with the mechanical mixer and their maximum capacity is 50g. This means the total mixture must weigh 50 g maximum.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

1) 2) 3) 4)

Put an empty and clean single use plastic cup on the precision scale- Tare the scale so it displays 0. Add the base PDMS (max 40g) and write down the value. Tare the scale so it displays 0- Using a pipette, add the catalyst (max 4g) to reach the ratio value 10:1 Place the cup in the mixing machine and adjust the revolution balance dial according to the total weight of the cup.

Caution: adapter weight of 115g to be added to the weight of your cup 5) Use the mechanical mixer to correctly homogenize the mixture (Program 1)

o Mixing: 1min @ 2000rpm o Defoaming: 2min @ 2200rpm 6) Clean the scale well before switching it off and clean the product bottles

2.2.3

Pouring Spin coating

Pour the PDMS mixture over the passivated mold placed in a Petri dish or plastic disposable dish. The interior of that dish should be protected with aluminum foil. 1) Be careful not to create bubbles while pouring the bubbles no bubbles mixture (proceed slowly). 2) The mixture is then degassed in the desiccator to remove any remaining entrapped bubbles. If large bubbles form at the surface, vent vacuum slowly so the mixture does not foam out. Put it back under vacuum until no bubbles are visible. This also improves the filling of small structures

2.2.4

Baking Curing

PDMS can cure without heating in ~24 hours. To decrease cure time, put the Petri dish in an oven for 1 hour at ~80C. Curing time depends on temperature and on the thickness of PDMS. After curing, the wafer is stable and can be stored for months if necessary. To save time, we provide you an already cured PDMS.

80 C

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

2.2.5

Alignment - Demolding Creating access ports by punching

After cooling, the PDMS is easily peeled off and cut. Use adequate tools to perform it (tweezers, razor blades). 1) 2) Cut the PDMS into the desired shape. DO NOT damage the device network! Create access ports using the manual hole punching machine fitted with a light source and a video camera. Alignment of PDMS samples with other glass/PDMS/silicon pieces can be done using the binocular.

hole punching machine

binocular

2.2.6

Surface activation for bonding

PDMS can be bonded to glass, silicon and itself using oxygen plasma surface activation. PDMS is hydrophobic, with a low energy and non-reactive surface. It is therefore difficult to bond it with other surfaces. By exposing PDMS PDMS to oxygen plasma, its surface becomes hydrophilic Plasma and more reactive. This results in irreversible bonding when it contacts glass, silicon, or even another PDMS glass coverslip piece that was exposed to the same oxygen plasma. Contact should be made quickly after plasma exposition because the PDMS surface will undergo reconstitution to its hydrophobic and non-reactive state within hours. A fine tuning of the oxygen plasma is necessary: a too long exposure will create too many Si-OH sites resulting in a non-sticking silica layer. A too short exposure will not create enough Si-OH sites for good bonding. 100 W, 0.3 torr and 6 sec are suggested as parameters. The bonding is accelerated if a post-bake is then performed.

2.3 Integration of Microuidics and Microscopy 2.3.1 Set up the pressure controller
The fluid flow through the device is controlled with a pressure controller, rather than a direct control of the flow rate. This means that the actual flow rates will be a function of the tubing length and diameter, the relative height of the different components, and the pressures. The needed pressures will therefore be slightly different for each time the experiment is set up. The MFCS controller needs a 10 minute warm up period. It should be turned on and warming up while the rest of the components are prepared. 1) 2) 3) Turn on the controller (power switch on the back). Open the MFCS_4C software. Press the green button on the front of the controller the warm up timer should begin counting down.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

Pressure channels

Figure 8.MFCS controller and software interface

2.3.2

Set up the tubing

While the controller is warming up, prepare the inlet and outlet tubing pieces. Minimizing the overall length of the tubing used will allow for the use of lower pressures and will also help with the flow stability of the system. 1) 2) 3) 4) Use equal length tubing for both inlets and equal length tubing for both outlets. Small pieces of steel adaptor tubing are used to couple the inlet and outlet tubing into the device. The adaptors will press-fit into the 0.02 ID Tygon tubing and also into the cored holes in the PDMS. Use the shortest length Tygon tubing that will reach from the device inlets to the sample tubes (fluiwells), leaving some room to move the device around on the microscope stage. The sample end of the inlet tubing will fit through the ferrule on the top of the sample tube in the pressure controller. The ferrule should be screwed down tightly to get the best pressure control. The end of the tubing should sit near the bottom of the sample tube. Use very short pieces of Tygon tubing for the outlets (~10 cm). Arrange the end of the outlet tubing so that the flow can spill into a suitable dish, e.g. a petri dish as shown in Figure 9. Make sure that the sample tubes are kept at the same height as the device.
Inlet 1 Media
Inlet 1 Media

5) 6) 7)

Outlet 1

Outlet 2

Inlet 2 Cells/beads

WASTE container

Outlet 1

Outlet 2 Inlet 2 Cells/beads

Figure 9. Tubing connection

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

2.3.3
1)

Sample preparation
Prepare a bead solution by adding some of the bead stock solution into buffer. A good bead concentration is at least 10L of 1 % bead stock solution per mL of buffer. Shake vigorously beforehand the bead solution anytime you handle it. Add about 2 mL of bead solution to a sample tube, and attach the sample tube to the appropriate channel in the sample holder. Fill another sample tube with buffer. Attach the sample tube to the appropriate channel in the sample holder. Make sure all components are sealed tightly.

2) 3) 4)

2.3.4 Microscopy
You will use the Olympus IX 81 inverted compound microscope. Please refer to the Microscopy/Koehler/dark field illumination section of the master handout to perform this step. Aside from initial calibration and occasional high-power measurements, you will find the 20x objective and darkfield illumination most useful. 1) Set up the Kogler illumination 2) Then, set the dark field illumination

2.4 Run the sample

In general, the waste outlets should always be at a lower pressure relative to the media and cell inlets. Since the outlets are not connected to the pressure controller, a positive pressure on the inlets will satisfy this. 1) To control pressures, the direct control button needs to be clicked. 2) The pressures in each channel can be controlled with the appropriate slider, or by entering the

value in the requested pressure box. You dont need to change any of the other control parameters. 3) The flow rate through the device to observe particle motion will be much lower than the flow rate needed to fill the inlet tubing in a reasonable time. The pressure controller has two channels with a range from 0-25 mBar and two channels with a range from 0-1000 mBar. Therefore, the inlet tubing should first be connected to the high pressure channels to fill the inlet tubing quickly, and then switched to the low pressure channels.
4) 5) 6) 7) 8) 9) Use channels 3 and 4 to fill the device with buffer. Make sure the tubing from the controller to the sample holder is connected from the correct channel to the correct sample. At pressures of around 50 100 mBar, filling the device will only take a minute or so. While fluid is flowing, inspect the device under the microscope to see if there are a significant amount of bubbles still in the device. If so, let the buffer run for a while longer at high pressure. Once the device is filled with fluid, turn the requested pressures to 0. You should be able to see beads in the channel when the fluid motion is stopped. Switch to controlling the pressure through channels 1 and 2 by changing the tubing connections at the controller. When running at low pressures to observe particle motion, the pressure difference between the media and cell/waste ports will be rather small to get flow from both into the waste outlets if one is too high, it will cause backflow into the other. The difference between the two is likely to be less than 1 mBar. Final

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

working pressures will be in the range of 2 - 10 mBar. At the lower pressures, the fluid motion will be slow enough to track the particle motion through the main channel. Only a few beads will enter the traps most will flow in main section. A good way to observe beads flowing through the traps is to use the 40X objective focused on a trap, with a higher pressure setting so that more beads are passing per time period.

2.5 Viewing / Tracking Particles in Device Geometry

Now that the device has been contacted and flow regulated, and the microscope has been fully configured, we are now ready to take data. 1) Use 2.5m beads sample to establish the pixel size of your camera 2) Turn on the Andor camera 3) Open Andor camera software called Solis 4) Turn the switch on the microscope to send an image to CCD camera 5) Click on the movie camera icon to get a live image from your sample 6) Set up the exposure time to 0.05s *by pressing exposure button (Figure 12), 7) To take pixel calibration image open in the main menu acquisition, under setup CCD, select c Single enter following values, exposure time 0.01-0.075s, next under Setup acquisition open binning to 512-512 pixels , you can move binning box to the region around your 2.5m bead press Ok and close Acquisition menu 8) Press Record and save image as sif file 9) Now we are ready to collect movies for your analysis session 10) To setup your movies, exposure time t , kinetic series length (number of frames in your movies ) open in the main menu acquisition, under setup CCD, select Kinetic series enter following values, exposure time 0.01-0.075s, kinetic series length 500, next under Setup acquisition if necessary open binning to 512512 pixels , you can move binning box to the region containing the most beads. Mark in your notebook the values you entered. Otherwise note in the notebook that you have take full images (1392*1040) 11) Press record 12) Save files as sif . Collect all necessary data and save them in your folder 13) Repeat this for several bead speeds. 14) Once you have finished data collection, you will need to convert all sif. files in raw files. You can do it file by file or using a batch conversion option in File menu (Main Menu). Make sure that you convert it in 16 bit unsigned integer (with range 0-65322). This is format required for the analysis session.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

Live

Record

Exposure

Figure 10. Andor Solis program for data acquisition. Bright field image of device and 1m beads.

2.6 Epifluorescence
Before starting, turn on the Mercury LAMP controller. We have only one filter cube set suitable or imaging of FITC labeled beads and GFP labeled bacteria. Locate its position (out of 6 possible) and open the filter cube shutter. If you observe bright blue light as shown on Figure 11, you have located it!
Figure 11. Epifluorescence

1) Now you can close the shutter and put light protection on the microscope body. 2) Use brightfield first to locate your specimen. 3) Switch to the Mercury lamp as the light source

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

4) Open the filter cube shutter 5) Make sure that sample is uniformly illuminated (if not open the diaphragm located close to the Mercury lamp) 6) Repeat the same measurements as in bright filed (for imageJ analysis fluorescence movies are easier to analyze!) Steps 6-14 are same maybe you will need to adjust exposure time Note. When not viewing the specimen, close the fluorescence shutter (push the shutter slider in) to minimize photobleaching of the specimen. Depending on the objective size you should observe something similar to images shown below!
a) b)

Figure 12. Fluorescence images of the device and beads using in a) 5 X in b) 40 x objective

7) Now stop running beads and try to flush only medium (water through the channel) this should remove
most of the beads from channels and leave the one in the traps.

8) Take fluorescence images of 5 traps! 3. DATA ANALYSIS

3.1 Calibration (done by TA beforehand)
1) 2) Open in ImageJ your dark filed image of 2.5m beads taken for bin size of 512*512 pixels or 1392*1040 pixels. Use a line tool in ImageJ toolbox to draw a line across the selected bead. Below the Image J toolbox you will notice x,y coordinates of your line together with the angle and the length of the drawn line. Make sure to draw the line straight across the bead diameter. Next open Analyze\Set Scale from File menu where you enter the length of 2.5m bead as known distance. It will calculate the pixel aspect ratio of either 1 (512*512) or 1.33 (1392*1040). Use these parameters to set a scale on all movies that you will be processing.

3)

Make sure you have used same objective and same binning! 3.2 Particle Tracking
To obtain single particle trajectories from recorded movies you will need to use Particle Detector and Tracker which is an ImageJ Plugin for particles detection and tracking from digital videos. The plugin implements the feature point detection and tracking algorithm as described in recent publication by Sbalzarini et al.6 This plugin presents an easy-to-use, computationally efficient, two-dimensional, feature point-

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

tracking tool for the automated detection and analysis of particle trajectories as recorded by video imaging in cell biology. The tracking process requires no apriori mathematical modeling of the motion, it is self-initializing, it discriminates spurious detections, and it can handle temporary occlusion as well as particle appearance and disappearance from the image region. The plugin is well suited for video imaging in cell biology relying on lowintensity fluorescence microscopy. It allows the user to visualize and analyze the detected particles and found trajectories in various ways: i) Preview and save detected particles for separate analysis; ii) Global non progressive view on all trajectories; iii) Focused progressive view on individually selected trajectory and iv) Focused progressive view on trajectories in an area of interest. It also allows the user to find trajectories from uploaded particles position and information text files and then to plot particles parameters vs. time - along a trajectory.

3.2.1 File opening

1) Before the plugin can be started you must open an image sequence or a movie in ImageJ. For opening your saved movie use the Import\ Raw from the File menu. You should input following parameters as indicated in Figure 13. (Check your lab notes for number of frames and binning size). Upon file import you should obtain video sequence of your moving beads.

Figure 13. Import parameters.

2) 3) 4)

Next you need to improve contrast and adapt your movie so that it can be treated with ParticleTracker plugin. To do so use the Image\Type\ 8 bit option from File menu. Next you need to increase contrast you will do it by using Process\Enhance Contrast option from File menu. It is safe to select 0.1%saturated pixels under Use Stack Histogram. To filter out noise use Process\Filter\Gaussian blur option from File menu. Again safe sigma value to use is 1.2. Before applying this filtering you can preview your movie.

Q8. What is going on when your sigma is larger than 3?

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

3.2.2 Plugin start

5) Now, that the movie is open and compatible with the plugging, you can start the plugin by selecting ParticleTracker from the Plugins -\ Particle Detector & Tracker menu. After starting the plugin, a dialog screen is displayed. The dialog has two parts Particle Detection and Particle Linking.

Figure 14. Parameters for better movie quality.

Particle Detection: This part of the dialog allows you to adjust parameters relevant to the particle detection (feature point detection) part of the algorithm Preview the detected particles: in each frame according to the parameters. This options offers assistance in choosing good values for the parameters. Save the detected particles according to the parameters for all frames. The parameters relevant for detection are: Radius: Approximate radius of the particles in the images in units of pixels. The value should be slightly larger than the visible particle radius, but smaller than the smallest inter-particle separation. Cutoff: The score cut-off for the non-particle discrimination Percentile: The percentile (r) that determines which bright pixels are accepted as Particles. All local maxima in the upper rth percentile of the image intensity distribution are considered candidate Particles. Unit: percent (%).

6)

Clicking on the Preview Detected button will circle the detected particles in the current frame according to the parameters currently set. To view the detected particles in other frames use the slider placed under the Preview Detected button. You can adjust the parameters and check how it affects the detection by clicking again on Preview Detected. Depending on the size of your particles and movie quality you will need to play with parameters.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

Note that very rarely you detect all particles in the field of view mostly due to the fact that they quickly go out of focus 7) To start on 2.5m beads, enter these parameters: radius = 6, cutoff = 0, percentile = 0.4 and click on preview detected. Check the detected particles at the next frames by using the slider in the dialog menu. With radius of 5 they are rightly detected as 2 separate particles. If you have any doubt they are 2 separate particles you can look at the 3rd frame. Change the radius to 10 and click the preview button. With this parameter; the algorithm wrongfully detects them as one particle since they are both within the radius of 10 pixels. Try other values for the radius parameter. Go back to these parameters: radius = 5, cutoff = 0, percentile = 0.4 and click on preview detected. It is obvious that there are more 'real' particles in the image that were not detected. Notice that the detected particles are much brighter then the ones not detected. Since the score cut-off is set to zero, we can rightfully assume that increasing the percentile of particle intensity taken will make the algorithm detect more particles (with lower intensity). The higher the number in the percentile field - the more particles will be detected. Try setting the percentile value to 2. After clicking the preview button, you will see that much more particles are detected, in fact too many particles - you will need to find the right balance (for our dark filed movies between 0.3-0.7 )

8)

There is no right and wrong here - it is possible that the original percentile = 0.1 will be more suitable even with this film, if for example only very high intensity particles are of interest.

Figure 15. Parameters for particle detection. On the left panel with default values. In the right movie with particles identified using following parameters. radius = 5, cutoff = 0, percentile = 0.4

3.2.3 Viewing the results

9) After setting the parameters for the detection (we will go with radius = 5, cutoff = 0, percentile = 0.6) you should set the particle linking parameters. The parameters relevant for linking are: Displacement: The maximum number of pixels a particle is allowed to move between two succeeding frames Link Range: The number of subsequent frames that is taken into account to determine the optimal correspondence matching.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

10) These parameters can also be very different from one movie to the other and can also be modified after viewing the initial results. Put following initial guess for the displacement=5 and link range =3.You can now go ahead with the linking by clicking OK. 11) After completing the particle tracking, the result window will be displayed. Click the Visualize all Trajectories button to view all the found trajectories. 12) Window displays an overview of all trajectories found. It cannot be saved! It is usually hard to make sense of so much information. One way to reduce the displayed trajectories is to filter short trajectories. Click on the Filter Options button to filter out trajectories under a given length. Enter 75 and click OK. (Be careful, if you select to long length you might end up with very few trajectories and lose information!). 13) Select a trajectory by clicking it once with the mouse left button. A rectangle surrounding the selected trajectory appears and the number of this trajectory will be displayed on the trajectory column of the results window. 14) Now that a specific trajectory is selected, you focus on it to get its information. Click on Selected Trajectory Info button. The information about this trajectory will be displayed in the results window. 15) Click on the Focus on Selected Trajectory button - a new window with a focused view of this trajectory is displayed. This view can be saved with the trajectory animation through the File menu of ImageJ. Look at the focused view and compare it to the overview window - in the focused view only the selected trajectory is displayed. 16) Finally you can save the data by pressing Save Full report. Repeat particle tracking for all 3 experimental conditions measured in the first part of the practical work (2 different speeds).

3.3 Matlab analysis Now when you have obtained single particle tracks for two different speeds by using provided matlab code you can: 1) Plot trajectories of certain length (not shorter than 50 frames) 2) Calculate speed (mark the exposure time) 3) Find and plot MSD

3.4 Particle analysis (If you have enough time)

The goal of this part is to count and determine the size distribution of trapped fluorescent beads. Particle counting can be done automatically if the specimen lends itself to it, i.e. the individual particles can touch but not too much! If automatic particle counting cannot be done, ImageJ can facilitate manual counting with the Point Picker or Cell counter plugin.

3.4.1 Automatic Particle counting

The biggest issue is one referred to as segmentation which is to distinguish the object from the background. Once the objects have been successfully segmented, they can then be analysed.

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3.4.2 Loading an image into the ImageJ program

1) 2) Open the ImageJ program. Go to File/Open. Select the picture that you would like to analyze. Note: It may be the case that you need to convert the image to a JPEG or analogous format. To do this, simply open the picture with Preview and save the picture as a JPEG using Save As, then use the toggle to select the format to JPEG. 3) You should now be able to see the image that you want to analyze. Optional: If you would like to know the actual areas of objects on the screen, say the areas of a collection of cells, and then knowing units is a must. ImageJ does this with ease: 4) On the ImageJ Tool Bar, select the straight-line icon. (This is on the same tool bar as a square, an oval, etc. Its the same tool bar that opens up with ImageJ.) 5) Using the straight-line tool, use the cursor to mark the length of any object on the picture that you know the length of. 6) On the top tool bar, select Analyze. 7) Scroll down to Set Scale. 8) Fill in Known Distance to the length of the object that you are measuring. This allows ImageJ to set up a pixel to distance ratio that allows area to be expressed in the appropriate units. 9) Fill in the units of measurement. Any units of distance should work: cm, mm, microns, etc. 10) Click Global. 11) Click Okay. 12) To check that you have indeed set the scale, use the line tool again and measure another object. Select Analyze and then select Measure. A window should pop up that displays the length of the object you just measured.

3.4.3 Particle analysis

1) 2) 3) Click on the image in the ImageJ window. On the top tool bar select Image. Select Type. Select 8-bit. This converts the image into a format that makes analysis possible. You should now see that your image is no longer in color. 4) If necessary crop only image part that contains trapped particles 5) You can do that by drawing the rectangle and by cropping the image Ctrl +Shift+X 6) On the top tool bar select Image. 7) Duplicate Image 8) On the top tool bar select Image. 9) Select Adjust, the select Threshold. 10) This step should have turned all of the objects of interest Red. (adjust scrollbar ) and then clicking on Apply once you are satisfied with the selection 11) This will turn your image in binary image (if you need you can fill holes by pressing Process-Binary-Fill Holes.) 12) If two particles are joined together, you can use processing filters such as Process Binary Watershed to separate them.

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ADVANCED BIOENGINEERING METHODS LABORATORY MICROFLUIDICS LAB ON CHIP Aleksandra Radenovic

13) On the top tool bar, select Analyze. 14) Select Analyze Particles. 15) A window will pop up. Under Size (in the units you specified), give the area that you want to analyze a lower bound. So if you wanted a minimum of say 10m2, you would write 10-Infinity in the box. Click Display Results and dont click any of the other boxes. All other boxes should be clear of check marks. 16) In the Toggle Menu, select Outlines. 17) Click Okay. 18) Two windows should have popped up. One with the areas of the objects listed in the units you specified and another window with the objects outlined with numbers inside their outlines. Each number with an area corresponds to the area of the object with that number in it. 19) Results will be saved as excel file.

3.4.4 Statistics
1) 2) 3) 4) 5) 6) 7) If you would like a distribution of the areas, click on your image again. The objects of interest should still be in red. On the top tool bar, select Analyze. Select Distribution. Unselect Automatic Binning. Write in the number of bins that you want and what area range to consider. Click Okay. A window should come up that gives you all necessary statistics for the areas of the objects in your picture and their distribution in a bar format.

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3.5 Questions
Q9. What is the average velocity of the moving beads? In which section of the channel does the interface move the fastest for a given applied pressure? Compute according to the Error Propagation Handout the standard deviation of the average velocity. Q10. For a given applied pressure, how will the fluid speed vary in the differently sized channels (as observable) by looking at the motion of the beads? Why? Q11. What is an average number of trapped beads? Can you suggest how to increase the number of trapped beads? Q12. Propose one biological application for a Lab on the chip device.

4. REFERENCE
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16 17 18

Groisman, A. et al. A microfluidic chemostat for experiments with bacterial and yeast cells. Nature Methods 2, 685-689, doi:Doi 10.1038/Nmeth784 (2005). Balagadde, F. K., You, L. C., Hansen, C. L., Arnold, F. H. & Quake, S. R. Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science 309, 137-140, doi:DOI 10.1126/science.1109173 (2005). Cookson, S., Ostroff, N., Pang, W. L., Volfson, D. & Hasty, J. Monitoring dynamics of single-cell gene expression over multiple cell cycles. Mol Syst Biol, -, doi:Artn 2005.0024 Doi 10.1038/Msb4100032 (2005).

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