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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.
TABLE OF CONTENTS
1
Theory............................................................................................................................................. 3
1.1
1.2
1.3
1.4
2.2
2.3
2.4
2.5
2.6
Epifluorescence ..................................................................................................................... 18
Calibration ............................................................................................................................. 19
3.2
3.3
3.4
3.5
Questions ............................................................................................................................... 26
References .................................................................................................................................... 26
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
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.
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.
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.
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.
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
Figure 4. Schematic of microuidic device fabrication using soft lithography (adapted from Ref. 8)
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)
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.
Q1. How does the laminar flow help microfluidic design? Why?
Q4. For what are the hooks between media input and waste outputs useful?
Q6. Define low Reynolds number. Typical E.coli (2.0m long and 0.5m in diameter) is characterized
by low or high Reynolds number?
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
10
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.
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)
2.2.1
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.
11
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
Glass
receptacle
Desiccator
3)
4)
5)
6)
7)
8)
15 min
12
1)
2)
3)
4)
2.2.3
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.
13
80 C
2.2.5
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
14
Pressure channels
2.3.2
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)
5)
6)
7)
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
Outlet 1
Outlet 2
Inlet 2 Cells/beads
WASTE container
Outlet 1
Outlet 2
Inlet 2 Cells/beads
15
2.3.3
1)
2)
3)
4)
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 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
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
16
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.
17
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
18
a)
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.
Open in ImageJ your dark filed image of 2.5m beads taken for bin size of
512*512 pixels or 1392*1040 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 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.
3)
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.
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-
19
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.
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.
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 Image\Type\ 8 bit option from File menu.
3)
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.
4)
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.
20
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.
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.
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.
21
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.
8)
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 )
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
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.
22
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).
23
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.
24
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.
25
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
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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preparation and electrophoretic analysis. Current Opinion in Biotechnology 14, 42-50, doi:Doi
10.1016/S0958-1669(02)00004-6 (2003).
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10.1002/elps.200305629 (2003).
Groisman, A., Enzelberger, M. & Quake, S. R. Microfluidic memory and control devices. Science 300,
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