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ADVANCED BIOENGINEERING METHODS LABORATORY

MICROFLUIDICS LAB ON CHIP

Aleksandra Radenovic

1


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 defined 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




2

TABLE OF CONTENTS

1 Theory 3

11 Basic Principles of Microfluidics 4

12 Device Fabrication 5

13 Questions (Theory) 8

14 Integration of Microfluidics and Microscopy 9

2 Practical work 11

21 Material requirements 11

22 PDMS silicon mold 11


24 Run the sample 16

25 Viewing Tracking Particles in Device Geometry 17

26 Epifluorescence 18

3 Data analysis 19

31 Calibration 19

32 Particle Tracking 19

33 Matlab analysis 23

34 Particle analysis 23

35 Questions 26

4 References 26




3

1 THEORY






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 fluid channels on the micron scale known as microfluidic

devices The goal of this exercise is to fabricate and use such a microfluidic 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 microfluidics 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

Microfluidics involve the handling and manipulation of very small fluid 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 fluid viscosity not inertia dominates fluid behavior at this scale microfluidic flow is laminar ensuring

that the system does not include turbulent flows which would be detrimental for observing cellular behavior under

high magnification

Lately microfluidic ldquolab-on-a-chiprdquo 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 microfluidic circuits analogous to their electrical

counterparts which aim to support large-scale multi-parameter analysis in parallel Recent applications of

microfluidics in biotechnology include DNA amplification purification separation 2 and sequencing

3 large-scale

proteomic analysis4 development of memory storage devices

5 cell sorting

6 and single-cell gene expression

profiling

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




4

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

11 Basic Principles of Microfluidics

111 Reynolds number The flow of a fluid through a microfluidic channel can be characterized by the Reynolds number defined as

equation (11)

avg

e

L VR

(11)

Where L is the most relevant length scale micro is the viscosity ρ is the fluid density and Vavg is the average

velocity of the flow For many microchannels L is equal to 4AP 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 ndash 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

112 Poiseuillersquos 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 (12)

and (12)

Where Δp 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 η is the dynamic fluid viscosity and x the distance in direction of flow

113 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 2a)

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 2a)

Δp=8μLQ

πr4

R=8ηΔx

πr4




5

114 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 2b) 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 2c) In closed

channels the velocity along the centerline of the channel is 50 of the velocity at the walls

a b

c

Figure 2 a) Velocity profile in a microchannel with aspect ratio 25 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

12 Device Fabrication

121 Photolithography

In recent years soft lithography has become the preferred method for fabricating microfluidic 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 first 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 fleshed out using engineering approaches Using the

laws of fluid dynamics under the condition of low Reynolds number for microvolume flow fluid channel

resistances are calculated and modified to satisfy desired driving pressures and flow rates Following fine-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 film 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




6

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 specified

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 final result

is a positive relief of photoresist on the silicon wafer known as a ldquomaster moldrdquo whose topology precisely

reflects 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 final 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 first 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 fluid reservoirs and cleaned

using Scotch tape and methanol In the final step the feature sides of the devices along with opposing coverslip

surfaces are briefly treated with low power oxygen plasma This process ldquoactivatesrdquo 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 fluid channels in the PDMS are sealed against the flat coverslip surface to form microchannels internally

connecting the device fluidic ports These finished devices mark completion of the replica molding step of soft

lithography




7

or create chrome mask

Your TArsquos prepared wafers beforehand

Concept copied form

Danino et al Nature 463 326-330 (2010)

Your will fabricate microfluidic device

from this point

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

122 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 microfluidic 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)




8

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 microfluidics field has produced several examples of complex devices with hugely parallel

active channel structures for high-throughput cell analysis In approaching years the fundamental benefits 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

13 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 Ecoli (20μm long and 05μm in diameter) is characterized

by low or high Reynolds number

Q7 How are fluidic resistance and channel width related




9

14 Integration of Microfluidics and Microscopy

Microfluidics 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 andor chemical gradients across the cell development volume Another recently demonstrated strength of

microfluidics 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 microfluidics 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 microfluidic network enabling the

passive and gentle separation of a single cell from bulk suspension14

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 microfluidic platform for long-term cell culture

studies spanning the entire differentiation process of mammalian cells15

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 microfluidic chip-based formats In continually providing fresh

nutrients and removing cellular waste to support exponential growth the microfluidic 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 microfluidic chemostat for culturing bacterial and yeast

cells in an array of shallow microscopic chambers with support for dynamically-defined media16

Similarly a

recent implementation of a microfluidic 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 ldquopopulation controlrdquo circuit that regulates cell

density through a feedback mechanism based on quorum sensing

A final implementation of the chemostat design was utilized to precisely control and constrain exponential

growth of the yeast Scerevisiae 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




10

141 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 non-

fluorescing 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 25 microm PeakFlowtrade 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 PeakFlowtrade 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 cytometer‟s 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




11

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

142Filters 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 ie the

wavelengths that illuminate the sample The red (2) curve shows the emission ie the wavelengths that are shown

to the viewer

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

2 PRACTICAL WORK

21 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 ovenhot plate oxygen plasma

Products Sylgard 184 silicone base Sylgard curing agent silanizing agent (TMCS

Chlorotrimethylsilane 33014 from sigma)

22 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 TA‟s prepared molds and they are in the AMBL marked wafer

holder

221 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




12

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)

Note If TMCS bottle is not in the glass desiccator

fetch it in the ldquosolventrdquo cabinet located on the right

side of the wet bench

TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes

3) Remove any dust on the surface of the mold using a

nitrogen gun

4) Place the siliconSU8 mold in this very same desiccator

5) Close the desiccator and place it under vacuum (this

causes the TMCS to evaporate and to form a passivation

layer on the mold surface)

6) Close well the TMCS bottle (use tape also) Fill-in the

ldquochemicals follow-uprdquo document 7) When desired time is reached (~15min) vent the

desiccator DO NOT breath directly above the open

desiccator

8) Take your mold back put the TMCS bottle in the

desiccator and put it back under vacuum

15 min

222 Mixing ndash 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




13

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

223 Pouring ndash 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

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

224 Baking ndash Curing

PDMS can cure without heating in ~24 hours To decrease cure time

put the Petri dish in an oven for 1 hour at ~80degC 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 degC

1) Put an empty and clean single use plastic cup on

the precision scale- Tare the scale so it displays 0

2) Add the base PDMS (max 40g) and write down the

value

3) Tare the scale so it displays 0- Using a pipette add

the catalyst (max 4g) to reach the ratio value 101

4) 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

bubbles no bubbles




14

binocular

hole punching

machine

225 Alignment - Demolding ndash Creating access ports by punching

After cooling the PDMS is easily peeled off and cut Use adequate

tools to perform it (tweezers razor bladeshellip)

1) Cut the PDMS into the desired shape DO NOT damage the device

network

2) Create access ports using the manual hole punching machine fitted

with a light source and a video camera Alignment of PDMS

samples with other glassPDMSsilicon pieces can be done using the

binocular

226 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 to oxygen plasma its surface becomes hydrophilic

and more reactive This results in irreversible bonding

when it contacts glass silicon or even another PDMS

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 03 torr and 6 sec are suggested as parameters The bonding

is accelerated if a post-bake is then performed


231 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) Turn on the controller (power switch on the back)

2) Open the MFCS_4C software

3) Press the green button on the front of the controller ndash the warm up timer should begin counting down

Plasma

glass coverslip

PDMS




15

Pressure channels

Figure 8MFCS controller and software interface

232 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) Use equal length tubing for both inlets and equal length tubing for both outlets

2) 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 002rdquo ID Tygon tubing and also into the cored holes in the PDMS

3) 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

4) 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

5) Use very short pieces of Tygon tubing for the outlets (~10 cm)

6) Arrange the end of the outlet tubing so that the flow can spill into a suitable dish eg a petri dish as

shown in Figure 9

7) Make sure that the sample tubes are kept at the same height as the device

Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container

Figure 9 Tubing connection




16

233 Sample preparation

1) 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

2) Add about 2 mL of bead solution to a sample tube and attach the sample tube to the appropriate channel

in the sample holder

3) Fill another sample tube with buffer Attach the sample tube to the appropriate channel in the sample

holder

4) Make sure all components are sealed tightly

234 Microscopy

You will use the Olympus IX 81 inverted compound microscope Please refer to the MicroscopyKoehlerdark

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 dark-

field illumination most useful

1) Set up the Kogler illumination

2) Then set the dark field illumination

24 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 bdquodirect 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 bdquorequested pressure‟ box You don‟t 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) 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

5) At pressures of around 50 ndash 100 mBar filling the device will only take a minute or so

6) 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

7) 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

8) Switch to controlling the pressure through channels 1 and 2 by changing the tubing connections at the

controller

9) When running at low pressures to observe particle motion the pressure difference between the media and

cellwaste ports will be rather small to get flow from both into the waste outlets ndash 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




17

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

25 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 25m 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 005s 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 001-0075s next under Setup acquisition open binning to 512-512

pixels you can move binning box to the region around your 25m 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 001-0075s kinetic series length 500 next under Setup acquisition if necessary open binning to 512-

512 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 (13921040)

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




18

RecordLive Exposure

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

26 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




19

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

31 Calibration (done by TA beforehand)

1) Open in ImageJ your dark filed image of 25m beads taken for bin size of

512512 pixels or 13921040 pixels

2) Use a line tool in ImageJ toolbox to draw a line across the selected bead Below

the Image J toolbox you will notice xy 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

3) Next open AnalyzeSet Scale from File menu where you enter the length of

25m bead as known distance It will calculate the pixel aspect ratio of either 1

(512512) or 133 (13921040) Use these parameters to set a scale on all

movies that you will be processing

Make sure you have used same objective and same binning

32 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 al6 This plugin presents an easy-to-use computationally efficient two-dimensional feature point-




20

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 low-

intensity 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

321 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) Next you need to improve contrast and adapt your movie so that it can be treated with ParticleTracker

plugin To do so use the ImageType 8 bit option from File menu

3) Next you need to increase contrast you will do it by using ProcessEnhance Contrast option from File

menu It is safe to select 01saturated pixels under Use Stack Histogram

4) To filter out noise use ProcessFilterGaussian blur option from File menu Again safe sigma value to

use is 12 Before applying this filtering you can preview your movie

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




21

322 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 amp Tracker menu After starting the plugin a dialog

screen is displayed The dialog has two parts ldquoParticle Detectionrdquo and ldquoParticle Linkingrdquo

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




22

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 25m beads enter these parameters radius = 6 cutoff = 0 percentile = 04 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

8) Try other values for the radius parameter Go back to these parameters radius = 5 cutoff = 0 percentile =

04 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 03-07 )

There is no right and wrong here - it is possible that the original percentile = 01 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 = 04

323 Viewing the results

9) After setting the parameters for the detection (we will go with radius = 5 cutoff = 0 percentile = 06) 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




23

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 =3You 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)

33 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

34 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 ie the individual particles can touch

ndash but not too much If automatic particle counting cannot be done ImageJ can facilitate manual counting with the

ldquoPoint Pickerrdquo or ldquoCell counterrdquo plugin

341 Automatic Particle counting

The biggest issue is one referred to as ldquosegmentationrdquo which is to distinguish the object from the background

Once the objects have been successfully segmented they can then be analysed




24

342 Loading an image into the ImageJ program

1) Open the ImageJ program

2) Go to FileOpen 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 It‟s 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

343 Particle analysis

1) Click on the image in the ImageJ window

2) On the top tool bar select Image Select Type

3) 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


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 ndash Watershed to

separate them




25


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 don‟t 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

344 Statistics

1) If you would like a distribution of the areas click on your image again The objects of interest should still

be in red


3) Select Distribution

4) Unselect Automatic Binning

5) Write in the number of bins that you want and what area range to consider

6) Click Okay

7) 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




26

35 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

1 Unger M A Chou H P Thorsen T Scherer A amp Quake S R Monolithic microfabricated valves

and pumps by multilayer soft lithography Science 288 113-116 (2000) 2 Ashton R Padala C amp Kane R S Microfluidic separation of DNA Current Opinion in Biotechnology

14 497-504 doiDoi 101016S0958-1669(03)00113-7 (2003) 3 Paegel B M Blazej R G amp Mathies R A Microfluidic devices for DNA sequencing sample

preparation and electrophoretic analysis Current Opinion in Biotechnology 14 42-50 doiDoi 101016S0958-1669(02)00004-6 (2003)

4 Lion N et al Microfluidic systems in proteomics Electrophoresis 24 3533-3562 doiDOI 101002elps200305629 (2003)

5 Groisman A Enzelberger M amp Quake S R Microfluidic memory and control devices Science 300 955-958 (2003)

6 Huh D Gu W Kamotani Y Grotberg J B amp Takayama S Microfluidics for flow cytometric analysis of cells and particles Physiol Meas 26 R73-R98 doiDoi 1010880967-3334263R02 (2005)

7 Danino T Mondragon-Palomino O Tsimring L amp Hasty J A synchronized quorum of genetic clocks Nature 463 326-330 doiDoi 101038Nature08753 (2010)

8 Lin F et al Generation of dynamic temporal and spatial concentration gradients using microfluidic devices Lab on a Chip 4 164-167 doiDoi 101039B313600k (2004)

9 Dertinger S K W Chiu D T Jeon N L amp Whitesides G M Generation of gradients having complex shapes using microfluidic networks Anal Chem 73 1240-1246 (2001)

10 Mao H Yang T amp Cremer P S A microfluidic device with a linear temperature gradient for parallel and combinatorial measurements J Am Chem Soc 124 4432-4435 doija017625x [pii] (2002)

11 Hong J W Studer V Hang G Anderson W F amp Quake S R A nanoliter-scale nucleic acid processor with parallel architecture Nature Biotechnology 22 435-439 doiDoi 101038Nbt951 (2004)

12 Fu A Y Chou H P Spence C Arnold F H amp Quake S R An integrated microfabricated cell sorter Anal Chem 74 2451-2457 doiDoi 101021Ac0255330 (2002)

13 Balaban N Q Merrin J Chait R Kowalik L amp Leibler S Bacterial persistence as a phenotypic switch Science 305 1622-1625 doiDOI 101126science1099390 (2004)

14 Wheeler A R et al Microfluidic device for single-cell analysis Anal Chem 75 3581-3586 doiDoi 101021Ac0340758 (2003)

15 Tourovskaia A Figueroa-Masot X amp Folch A Differentiation-on-a-chip A microfluidic platform for long-term cell culture studies Lab on a Chip 5 14-19 doiDoi 101039B405719h (2005)




27

16 Groisman A et al A microfluidic chemostat for experiments with bacterial and yeast cells Nature Methods 2 685-689 doiDoi 101038Nmeth784 (2005)

17 Balagadde F K You L C Hansen C L Arnold F H amp Quake S R Long-term monitoring of bacteria undergoing programmed population control in a microchemostat Science 309 137-140 doiDOI 101126science1109173 (2005)

18 Cookson S Ostroff N Pang W L Volfson D amp Hasty J Monitoring dynamics of single-cell gene expression over multiple cell cycles Mol Syst Biol - doiArtn 20050024 Doi 101038Msb4100032 (2005)




2

TABLE OF CONTENTS

1 Theory 3

11 Basic Principles of Microfluidics 4

12 Device Fabrication 5

13 Questions (Theory) 8


2 Practical work 11

21 Material requirements 11

22 PDMS silicon mold 11


24 Run the sample 16

25 Viewing Tracking Particles in Device Geometry 17

26 Epifluorescence 18

3 Data analysis 19

31 Calibration 19

32 Particle Tracking 19

33 Matlab analysis 23

34 Particle analysis 23

35 Questions 26

4 References 26




3

1 THEORY





















high magnification






3 large-scale


5 cell sorting


profiling









4







equation (11)

avg

e

L VR

(11)












and (12)













Δp=8μLQ

πr4

R=8ηΔx

πr4




5










a b

c

























6


plate




shown in Figure 3



gene network7





















lithography




7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







3

1 THEORY





















high magnification






3 large-scale


5 cell sorting


profiling









4







equation (11)

avg

e

L VR

(11)












and (12)













Δp=8μLQ

πr4

R=8ηΔx

πr4




5










a b

c

























6


plate




shown in Figure 3



gene network7





















lithography




7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







4







equation (11)

avg

e

L VR

(11)












and (12)













Δp=8μLQ

πr4

R=8ηΔx

πr4




5










a b

c

























6


plate




shown in Figure 3



gene network7





















lithography




7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







5










a b

c

























6


plate




shown in Figure 3



gene network7





















lithography




7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







6


plate




shown in Figure 3



gene network7





















lithography




7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







7



Concept copied form



from this point


















8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








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18

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controller













19



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a) b)





3 DATA ANALYSIS





















20











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21

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22


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33 Matlab analysis


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the length of






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measured







color




7) Duplicate Image






Holes)


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25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







8























9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

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22


focus






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23










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results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







9










strategies










weeks









Similarly a























10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







10
















a) b)

Wavelenght (nm)

Spectrum




















11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

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Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







11






to the viewer


2 PRACTICAL WORK



aluminum foil









holder







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







12












TMCS

Wafer with

PDMS mold

Glass

receptacle

Desiccator

single use

pipettes


nitrogen gun








desiccator



15 min









13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

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Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







13























80 degC




value





weight of the cup


weight of your cup

bubbles no bubbles




14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







14

binocular

hole punching

machine





network




binocular



























Plasma

glass coverslip

PDMS




15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







15

Pressure channels















shown in Figure 9


Inlet 1 Media

Inlet 2 Cellsbeads

Outlet 1 Outlet 2

Outlet 1 Outlet 2

Inlet 2 Cellsbeads

Inlet 1 Media

WASTE container





16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







16








holder


234 Microscopy







24 Run the sample






parameters













controller







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







17

















menu








11) Press record









18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







18

RecordLive Exposure


26 Epifluorescence


controller













19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







19



lamp)






a) b)





3 DATA ANALYSIS





















20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







20











321 File opening
















21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







21

322 Plugin start















Unit percent ()









22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







22


focus






10 pixels

















frames






23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







23










information)



results window









33 Matlab analysis


code you can















24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







24













the length of






10) Click Global

11) Click Okay



measured







color




7) Duplicate Image






Holes)


separate them




25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







25









17) Click Okay





it


344 Statistics


be in red





6) Click Okay






26

35 Questions








4 REFERENCE




















27







26

35 Questions








4 REFERENCE




















27







27




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