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1_______________________________________________________________________________________________“NOTICE: this is the author’s version of a work that was accepted for publication in BioChip Journal. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version will be subsequently published in BioChip Journal, [Vol.7(4), pp.367‐374, (2013)].” _______________________________________________________________________________________
A Microfluidic Device for Partial Cell Separation and Deformability Assessment
Diana Pinho1, 2, Tomoko Yaginuma1 & Rui Lima*1, 2
1 Polytechnic Institute of Bragança, ESTiG/IPB, C. Sta. Apolonia, 5301-857 Bragança, Portugal. 2 CEFT, Faculdade de Engenharia da Universidade do Porto (FEUP), R. Dr. Roberto Frias, 4200-465 Porto, Portugal. * Correspondence and requests for materials should be addressed to R. Lima (ruimec@ipb.pt)
Abstract Blood flow in microcirculation shows several interesting phenomena that can be used to develop microfluidic devices for blood separation and analysis in continuous flow. In this study we present a novel continuous microfluidic device for partial extraction of red blood cells (RBCs) and subsequent measurement of RBC deformability. For this purpose, we use polydimethylsiloxane (PDMS) microchannels having different constrictions (25%, 50% and 75%) to investigate their effect on the cell-free layer (CFL) thickness and separation efficiency. By using a combination of image analysis techniques we are able to automatically measure the CFL width before and after an artificial constriction. The results suggest that the CFL width increases with enhancement of the constriction and contributes to partial cell separation. The subsequent measurements of RBCs deformation index reveal that the degree of deformation depends on the constriction geometries and hematocrit after the cell separation module. The proposed microfluidic device can be easily transformed into a simple, inexpensive and convenient clinical tool able to perform both RBC separation and deformability analysis in one single device. This would eliminate the need for external sample handling and thus reducing associated labor costs and potential human errors. Keywords: Biomicrofluidics, Microfluidic devices, Microcirculation, Blood on chips, Red blood cells, Cell separation, Cell deformability, Deformation index.
Introduction
Cell separation and identification are essential in a variety of biomedical applications including cell biology, diagnostic and therapeutic methods. Blood is a non-Newtonian fluid containing extremely rich amount of information about the physiological and pathological state of the human body. However, due to its complexity there are few accurate analysis methods. Most of the standard techniques used cell separation and sorting are often labor intensive or require additional external labels to identify cells.
Blood flow in microcirculation shows several interesting phenomena that can be used to develop microfluidic devices for blood separation and analysis in continuous flow. These phenomena include plasma skimming [1], cell-free layer (CFL) [2, 3, 4], leukocyte margination [5] and bifurcation law [6]. Recently, several researchers have used these effects and replicated in microfluidic systems. In microchannels RBCs, due to their deformability and lift forces, tend to be concentrated around the center of the microchannels while white blood cells (WBCs) and rigid RBCs (such as Malaria RBCs) tend to migrate to the CFL originated at regions close to the walls [1-5]. Bifurcation law [6] states that RBCs behavior, in microchannels with bifurcations, tends to be oriented to the wide microchannel. A number of microfluidic devices have been developed to take advantage of these natural hemodynamics phenomena. Shevkoplyas et al. [7] developed a microdevice to isolate WBCs from a blood sample by using the margination effect, whereas Hou et al. [8] have very recently proposed a biomimetic separation device to separate normal RBCs from malaria infected RBCs. Other researchers have found several advantages to control and manipulate blood flow in microfluidic devices. Fujiwara et al. [9] have found evidence that it is possible to create an artificial CFL under appropriate hemodynamic and geometrical conditions, and also the CFL thickness is strongly influenced by the RBC deformability.
2
Recently, Ishikawa et al. [10] and Leble et al. [11] have shown the existence of thin CFL in the centre of the microchannel, just downstream of a confluence. This phenomenon is due to the existence of a CFL in both inner walls and a consequent formation of a triangular CFL in the region of the confluence apex [10, 11]. Faivre et al. [12] and Sollier et al. [13] have demonstrated that the CFL could be enhanced by using a microchannel containing a constriction followed by sudden expansion to separate plasma from the whole in vitro blood. However, most of these studies aim at the complete extraction of cells from plasma, which is not the case of the present study.
The main objective of the proposed microfluidic device is to separate certain amount of RBCs from plasma and then measure the deformability of individual RBCs downstream in one single step. RBC deformability is important in a clinical sense as it is related to several diseases such as diabetes, malaria, as well as cardiovascular disorders [14]. These diseases are at times fatal and early detection is crucially preferable. In this sense, RBC deformability can be a new biomarker and the fast and easy measurement methods are required. Former studies on RBC deformability have introduced some methodologies such as micropipette aspiration and optical tweezers where basically RBCs are stretched and the pressure or force for their extension is measured [15, 16]. Whilst these studies have revealed useful RBCs mechanical properties, these methods reqire large amount of preparation and are very time consuming. For fast analysis of vast amount of blood samples, these traditional measurement methods are unlikely to be appropriate. Moreover, since the preceded cell separation process is needed, the microfluidic devices which are easy to manipulate their microchannel geometries are more suitable for our purposes. A typical microdevice for RBC deformation studies uses a microchannel having a shape of sudden constriction in order to elongate cells. Zhao et al. [17] used a straight microchannel with a sudden narrowing and expanding constriction. Lee et al. [18], Yaginuma et al. [19-20] and Faustino et al. [21], on the other hand, used microchannels with a hyperbolic shape contraction followed by a sudden expansion region. These microfluidic experiments showed high deformability of RBCs when travelling through a contraction region. However, those studies did not include the preceded cell separation process as they have solely performed deformability measurements after an additional sample preparation [19-21].
The proposed microfluidic device aims to obtain a CFL with a low enough RBC concentration to perform cell deformability measurements downstream the separation constriction. This study is divided in two main parts. Firstly, a simple microfluidic device with different constrictions (25%, 50% and 75%) is used to test the RBCs separation. Secondly, a more complex device, able to perform in a single step both RBC separation and deformability analysis, was tested. This first tentative to integrate in one single device both tasks of separation and deformation have shown not only the viability of this new clinical strategy but also new findings that will be crucial to optimize the design for this kind of microfluidic device.
Materials and methods Working fluids The working fluid used in this study was dextran 40 (Dx40) containing about 9% (i.e. Hematocrit, Hct = 9) by volume of human RBCs. More details can be found in Supplementary Materials. Microdevice geometries The microfluidic devices tested in this study were fabricated using a soft lithography technique and consist of two main parts: a cell separation region and a cell deformation region. The microchannel height was measured by a profilometer to be 51 m.
Fabrication of the microfluidic devices The polydimethylsiloxane (PDMS) rectangular microchannels were fabricated using a soft lithographic technique. A detailed description of the fabrication process can be found elsewhere [22, 23]. Briefly, the microchannel geometry was drawn using Autocad, and a high resolution photomask was manufactured. The solid master was then fabricated on a silicon wafer with an ultrathick photoresist (SU-8 50; Kayaku MicroChem, Japan). The PDMS prepolymer was prepared by mixing a comercial prepolymer and catalyzer (Silpot 184; Dow Corning, USA) at a weight ratio of 10:1. After the mixture was degassed under vacuum, the PDMS was poured into the SU-8 photo-resist master mold and cured by baking for about 2h at 70°C. Both master and PDMS were cooled to room
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esented in Finstream regiment is moreCFL . Addi
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igure 2, showion CFL . e pronounceditionally, thehich corrobo
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deformatione as parachuhis shape doewere often ob
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llowed in ty, two geoma smooth conthis examina
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due to the conigure 3a showmicrochanner stress causefrom an ellipalso the iman of RBCs wute shapes aes not have bserved, whi
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5
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Figure 4flow rate
Figure 5measureconfiden
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4. The differes and differe
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mentioning th 0.5 and r =low rates. Hockness (see Fre 5 shows th 0.5 this co
ng 27% of cnalysis approred that all th
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n rate of the expressed as
detail the effeonstriction aasing the rorders of maat the highes= 0.25 for loowever theseFigure 5). he separationonstriction ccells to the doach used to he white par
CFL upstreaion ratios (r
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agnitude greast ∆ corow flow ratee results show
n rate of cellscan efficientldeformation calculate the
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± standard de
tificial constrn of the flownhancement iater than ∆rresponds toe there is a tw evidence t
s flowing to tly separate region. This
e CFL thicknthe constrict
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utlet region neviation acco
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for r = 0.r = 0.25 antendency to that the const
the outlet regabout 73%
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number 2 (upording to a t-t
he CFL thicke results shoounced for r5 and r = 0.
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kness, Figureow clearly thr = 0.25 wh.75, respectiv
ndent of the fut tend to flaa strong imp
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6
different
tlet). The at a 95%
e 4 shows hat ∆
here ∆ vely. It is flow rate. attened to act in the
re 6), i. e., on 2 and the same
mages and bution of
the RBCwere ablconstrictshown aHence, wr = 0.5decided single stfeed Hctrecently,contractirate shouthe geomlocal Hc
Figure 6separatiom, respof deformThe heig
Figure 7contracti
Cs core to bele to estimattion with higa significant we believe th5 is by decreto use the coep both sepat to be abou, by using ions [20]. Inuld be arounmetrical paracts less than 1
6. Schematicon part. The pectively. Thmation meas
ght of the mic
7. Mean DI ions.
e 100%. Hente the separagher efficiendifference b
hat the most easing the geonstriction waration and dut 9%, the lo1% Hct we n order to a
nd 90% and ameter L2, to1%.
c drawing of dimensions
he different csurement parcrochannel i
values of tw
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have succechieve 1% Hconsequentl
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f the microfluof L , L , w
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wo deformatio
suring the CFpending on one with r
= 0.25 and ay to get a coarameter L2
to test the abof blood cellthe outlet 3
essfully meaHct with thely r must beon less than
uidic device.w , w , w ,
were used (ensions of w
on measurem
FL thicknessthe size of tr = 0.25. Ar = 0.75, tonsiderable d(see Figure
bility of the ps. Note that (deformatio
asured the Re proposed me less than 0100 µm, it w
. (a) the entirare 300 m,
= 75 mw and w are
ment module
s downstreamhe constricti
Although statthis is not thdifference be6). In the pr
proposed devfor r = 0.5 n region) is
RBC deformamicrofluidic d0.25. Alternawill be possi
re view and 100 m, 10
, 50 m and e 7 m and
es: (a) a sudd
m the constriion. As expetistical analy
he case for retween r = resent study
vice to perfor and by consaround 2.4%
mability in hydevice the s
atively, by deible to achie
close-up vie00 m, 80 m
25 m). (b)10 m, resp
den and (b)
7
iction we ected, the ysis have r = 0.5. 0.25 and we have
rm in one sidering a %. Very yperbolic eparation ecreasing
eve easily
ew of cell m and 20 the view
pectively.
a smooth
8
In terms of the deformation measurements, Figure 7 shows average DI values of ten RBCs flowing through the two different constriction regions. The maximum DI value is in both cases relatively the same, however, for the sudden contraction case (a) the maximum DI is located close to the constriction entrance whereas for the smooth case (b) the maximum DI is located around the middle of the constriction. In addition, in the constricted region in (a) RBCs tend to flow as parachute like shape and the DI values are not truly comparative in the sequence of cell behaviour throughout the deformation analysis region of the device. At least, the region for measurements has to be carefully selected for a meaningful comparison. On the other hand, the smooth contraction provides less deviation of DI results and indicates clearly an appropriate measurement region (x/Lc=0.5), i. e., middle of constriction. The majority of the cells are flowing with the same orientation at the centreline of the channel where the cells are under an extensional flow dominated regime and as a result they tend to elongate in y direction induced mainly by this extensional force. This stable state of the RBCs makes this geometry suitable to measure small changes of RBC deformability. More detailed studies on RBCs extensional flow effects can be found elsewhere [19-21].
As mentioned before, by using a constriction with r = 0.5, we have observed collisions and the overlap of neighbouring cells flowing within the measurement region. In the CS region, the Hct of the flowing fluid is reduced from 9% to ≈2.4% but ideally it needs to be less than that. Nevertheless, it is worth mentioning that by reducing Hct to less than 2% the number of RBCs to measure may not be large enough to obtain a significant statistical picture of the results. Therefore, an optimization of channel, i.e. change in depth, needs to be done in order to obtain the best representative results of the RBCs DI.
Conclusions The conventional microfluidic methods for measuring RBC deformability are often labor intensive and require additional sample modification and preparation. In this paper, we present a new, simple microfluidic device able to perform both RBCs separation and deformability assessment in one single step. In general, our results indicate that the proposed device can perform both operations (separation and deformation) successfully. The reported results show evidence that the constriction has a strong impact on the CFL thickness and consequently on separation rate. Moreover, the deformability results show clearly that most appropriate geometry to measure RBC deformability is the microchannel containing a smooth constriction region. In this kind of geometry due to the existence of a dominant extensional force the majority of the RBCs tend to flow with the same orientation. This stable performance of the RBCs may prove to be enough sensitive to detect small changes of RBC deformability and thus it may have the ability to diagnose early stage RBC related diseases such as diabetes, malaria and sickle cell anemia. Therefore, the integrated and simple continuous system operations make the proposed microfluidic device a potential diagnostic technique to be applied to both healthy cells and blood cell diseases. Acknowledgement The authors acknowledge the financial support provided by 2007 Global COE Program “Global Nano-Biomedical Engineering Education and Research Network”, Japan and grant-in-Aid for Science and Technology PTDC/SAU-BEB/108728/2008, PTDC/SAU-BEB/105650/2008, PTDC/EME-MFE/099109/2008, PTDC/SAU-ENB/116929/2010 and scholarship SFRH/BD/89077 /2012 from FCT (Science and Technology Foundation), COMPETE, QREN and European Union (FEDER). The authors are also very grateful to Dr. Mónica Oliveira (Strathclyde University), Professor Takuji Ishikawa and Professor Takami Yamaguchi (Tohoku University) for their suggestions and support to this research work.
References
[1] Caro, C., Pedley, T., Schroter, R. & Seed, W. The Mechanics of the Circulation. Oxford University Press, 1978.
[2] Lima, R. et al. Measurement of individual red blood cell motions under high hematocrit conditions using a confocal micro-PTV system. Ann Biomed Eng. 37, 1546-59, (2009).
[3] Fujiwara, H., et al. Red blood cell motions in high-hematocrit blood flowing through a stenosed microchannel. J Biomech 42, 838-843, (2009).
9
[4] Lima, R., et al. Axisymmetric polydimethysiloxane microchannels for in vitro hemodynamic studies. Biofabrication, 1: 1-7, (2009).
[5] Goldsmith, H., & Spain, S. Margination of leukocytes in blood flow through small tubes. Microvasc Res, 27, 2, 204-222, (1984).
[6] Yang, S., Undar, A., & Zahn, J.D. A microfluidic device for continuous, real time blood plasma separation. Lab Chip 6:871, (2006).
[7] Shevkoplyas, S.S., Yoshida, T., Munn, L.L., & Bitensky, M.W. Biomimetic Autoseparation of Leukocytes from Whole Blood in a Microfluidic Device. Anal Chem, 77, 933-937, (2005).
[8] Hou, H.W., et al. Deformability based cell margination - A simple microfluidic design for malaria-infected erythrocyte separation. Lab Chip, 10, 2605–2613, (2010).
[9] Fujiwara, H., et al. Red blood cell motions in high-hematocrit blood flowing through a stenosed microchannel. J Biomech 42, 838-843, (2009).
[10] Ishikawa, T., et al. Asymmetry of blood flow and cancer cell adhesion in a microchannel with symmetric bifurcation and confluence. Biomed Microdevices, 13, 159, (2011).
[11] Leble, V., et al. Asymmetry of red blood cell motions in a microchannel with a diverging and converging bifurcation. Biomicrofluidics, 5, 044120, (2011).
[12] Faivre, M., Abkarian, M., Bickraj, K. & Stone, H. Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma. Biorheology, 43, 147-159, (2006).
[13] Sollier, E., Cubizolles, M., Fouillet, Y. & Achard J. Fast and continuous plasma extraction from whole human blood based on expanding cell-free layer devices. Biomed Microdevices, 12, 485-97, (2010).
[14] Mokken, F.C., Kedaria, M., Henny, C.P., Hardeman, M.R., & Gelb, A.W. The clinical importance of erythtrocyte deformability, a hemorrheological parameter. Ann Hematol, 64, 113-122, (1992).
[15] Musielak, M. Red blood cell deformability measurement: Review of techniques. Clin Hemorheol Micro, 42, 47–64, (2009).
[16] Shiga, T., Maeda, N., & Kon, K. Erythrocyte rheology. Crit Rev Oncol Hemat, 10: 9-48, (1990).
[17] Zhao, R., et al. Microscopic investigation of erythrocyte deformation dynamics. Biorheology, 43(6), 747-765, (2006).
[18] Lee, S.S., Yim, Y., Ahn, K.H., & Lee, S.J. Extensional flow-based assessment of red blood cell deformability using hyperbolic converging microchannel. Biomed Microdevices, 11, 1021-1027, (2009).
[19] Yaginuma, T., Oliveira, M.S.N., Lima, R., Ishikawa, T., & Yamaguchi, T. Red blood cell deformation in flows through a PDMS hyperbolic microchannel, Proceedings of NSTI-Nanotech, 2 505-507, (2011).
[20] Yaginuma, T., Oliveira, M.S.N., Lima, R., Ishikawa, T., & Yamaguchi, T. Human red blood cell behavior under homogeneous extensional flow in a hyperbolic-shaped microchannel. Biomicrofluidics, 7:054110, (2013).
[21] Faustino V., Pinho D., Yaginuma T., R. Calhelha, M. Oliveira, I. Ferreira, Lima R., Flow of red blood cells suspensions through hyperbolic microcontractions. Visualization and simulation of complex flows in biomedical engineering. In: R.Lima, T. Ishikawa, Y. Imai & M. S. N. Oliveira (Eds), Springer, 2013 (in press).
[22] Lima, R., Wada, S., Tanaka, S., Takeda, M., Ishikawa, T., Tsubota, K., Imai, Y., Yamaguchi, T., In vitro blood flow in a rectangular PDMS microchannel: experimental observations using a confocal micro-PIV system. Biomedical Microdevices, 10: 153-167, 2008.
[23] Lima, R., et al. Microscale flow dynamics of red blood cells in microchannels: an experimental and numerical analysis. In: Tavares and Jorge (Eds) Computational Vision and Medical Image Processing: Recent Trends, Springer 19, 297-309, (2011).
[24] Abramoff, M., Magelhaes, P. & Ram, S. Image Processing with ImageJ. Biophotonics International, 7, 11, 36-42, (2004).
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Supplementary Materials
1. MATERIALS AND METHODS
1.1. Working fluids
The working fluid used in this study was dextran 40 (Dx40) containing about 9% (i.e. Hematocrit
(Hct = 9%). Briefly, blood was collected from a healthy adult volunteer and heparin was added in
order to prevent coagulation. The red blood cells (RBCs) were separated from bulk blood by
centrifugation and aspiration of the plasma and buffy coat. The RBCs were then washed twice with a
physiological saline solution and diluted with Dx40 to make up the required RBC concentration. All
blood samples were stored hermetically at 4ºC until the experiments were performed at controlled
temperature of approximately 37ºC.
1.2. Fabrication of the microfluidic devices
The polydimethylsiloxane (PDMS) rectangular microchannels were fabricated using a soft
lithographic technique. A detailed description of the fabrication process can be found elsewhere [22,
23]. Briefly, the microchannel geometry was drawn using Autocad, and a high resolution photomask
was manufactured. The solid master was then fabricated on a silicon wafer with an ultrathick
photoresist (SU-8 50; Kayaku MicroChem, Japan). The PDMS prepolymer was prepared by mixing a
comercial prepolymer and catalyzer (Silpot 184; Dow Corning, USA) at a weight ratio of 10:1. After
the mixture was degassed under vacuum, the PDMS was poured into the SU-8 photo-resist master
mold and cured by baking for about 2h at 70°C. Both master and PDMS were cooled to room
temperature and the PDMS was peeled from the master. The input/output ports are made by means of
micro-pipette tips. Finally, the PDMS was washed with ethanol and brought into contact with a clean
slide glass, where a reversible seal formed spontaneously.
1.3. Experimental set-up
The high-speed video microscopy system used in this study is shown in Figure 1. This system
consisted mainly by an inverted microscope (IX71; Olympus, Japan) combined with a high-speed
camera (Phantom v7.1; Vision Research, USA). All the microfluidic devices were placed on the stage
of the inverted microscope and by using a syringe pump (KD Scientific, USA), a constant pressure-
driven was maintained. Additionally, a thermo plate controller (Tokai Hit) was set to 37ºC in order to
have an environment close to in vivo conditions.
Figure devices.
1.4. Ima
Cell sepa
All imag
pixels, a
with the
L/min.
[24].
Figure 21 L/min
First, the
with ma
function
region c
converte
1. High-spe
age analysis
aration (CS)
ges were cap
at a rate of 80
flow of RBC
The record
2. Original imn.
e captured v
aximum inten
n, which resu
corresponds t
ed to binary i
ed video m
) microfluidi
ptured around
000 frames/s
Cs (halogen
ded images w
mage with th
videos were
nsity of all
ults in a sing
to RBCs cor
images with
icroscopy sy
ic device
d the middle
s and an expo
illumination
were transferr
he flow of RB
converted to
the images
le image hav
re. To obtain
thresholding
ystem used
e plane of the
osure time o
n) through a 2
rred to the co
BCs through
o a sequence
in the stack
ving a region
n quantitativ
g (see Figure
to test the
e microchann
f 0.125 ms. F
25% constric
omputer and
a 75% const
e of static im
k was select
n brighter th
ve measurem
3).
performance
nels with a re
Figure 2 sho
ction for a co
then evaluat
triction for a
mages (stack
ed by using
han the backg
ments the gre
e of the mic
esolution of
ows an origin
onstant flow
ated in Image
a constant flo
k). Then, eac
g ImageJ “Z
ground. This
ey scale ima
11
crofluidic
800304
nal image
w rate of 1
e J (NIH)
ow rate of
ch pixel’s
Z project”
s brighter
ages were
Figure 3The chan
Cell sepa
RBC de
contracti
the regio
flowing
Figure 4 Deforma
Deforma
where Lm
like shap
3. Binary imnnel dimensi
aration and
eformation a
ion region o
on. Ten cell
cells were m
4. Schematic
ation Index
ation Index (
major and Lmin
pe RBCs, we
mage obtaineions are: w1 =
deformation
analysis has
f the microc
s were selec
measured man
c diagram of
DI) is define
nor are the ma
e measured th
ed after imag= 100 m an
n (CSD) micr
been achiev
hannel (W5
cted for each
nually frame
RBC deform
ed as:
ajor and mino
he major and
ge processingnd w2 = 25 m
rofluidic dev
ved by mea
and W6 in Fi
h contraction
e by frame.
mation measu
or axis lengt
d minor axis
g and binarim.
vice
asuring RBC
igure 4), inc
ns, and majo
urement regio
hs of the RB
lengths as sh
zation using
C deformatio
luding upstre
or and mino
ons with deta
BC (see Figur
hown in Figu
g “Image J”
on index (D
ream and dow
or axis length
ailed dimens
re 5a). For p
ure 5b.
12
software.
DI) at the
wnstream
hs of the
sions.
arachute-
Figure 5measurethe majo The mea
(NIH) [2
The 2D
opened a
flowing
by the R
RBC’s a
it by the
found to
deviation
2. RESUFigure 6
measure
Figure 6side) for
Figure 7
device u
5. Schematicment of para
or and minor
asurements w
24]. We also
image cont
and this shad
direction (y)
RBC’s actual
actual velocit
e frame inter
o be suffici
ns were igno
ULTS 6 shows repr
ments obtain
6. Original br different art
7 shows a re
upstream the
c view of DIachute shapeaxes are def
were done m
calculated th
ains the flow
dow should
). To avoid t
velocity tim
ty, we measu
rval time. Th
iently low (
ored.
resentative o
ned at differe
blood flow vtificial constr
epresentative
separation re
I measuremee cells. The bfined.
manually wit
he length err
wing cell’s s
not be consi
this error the
mes the expos
ured the trav
he cell’s trav
(≪ 1µm) th
original imag
ent artificial c
isualization rictions (r =
e original im
egion with r
ents. (a) the box surround
th the assista
ror which mi
shadow whi
idered as the
e cell’s trave
sure time of
veling length
veling length
hroughout th
ges and corre
constrictions
images (left = 0.25, r = 0
mage of the
= 0.5.
measuremending the cell
ance of an i
ight be cause
ich is captur
e cell’s actua
ling length (
f the high spe
h of the cells
h values obt
he measured
espondent bi
s (r = 0.25, r
side) and co0.5 and r = 0
deformation
nt of ellipsoidefines the c
mage handli
ed by the cam
red when the
al length esp
(shadow) in y
eed camera. I
frame by fra
tained by the
d regions so
inary images
r = 0.5 and
orrespondent0.75).
region of th
id shape cellcenter of the
ing software
mera’s expos
e camera’s s
pecially in th
y-axis was c
In order to o
ame and then
ese calculati
o that these
s from in vit
r = 0.75).
t binary imag
he CSD mic
13
s. (b) the e cell and
e, ImageJ
sure time.
shutter is
he axis of
calculated
obtain the
n divided
ons were
possible
tro blood
ges (right
crofluidic
Figure 7containin
Figure 8
The Hct
the cell c
Figure 8containin
7. Blood flowng a sudden
8 shows a rep
at this regio
can be observ
8. Blood flowng a smooth
w visualizaticonstriction
presentative
on should be
ved at the na
w visualizaticonstriction
ion images omicrochanne
original ima
e also around
arrowest part
ion images omicrochann
of the deformel with a wid
age of the de
d 2.4%. In th
t of the contr
of the deformnel with a wid
mation regiondth of 7 m.
eformation r
his contractio
raction.
mation regiondth of 10 m
n of the CSD
egion with a
on, the maxi
n of the CSDm.
D microfluid
a smooth con
imum deform
D microfluid
14
dic device
ntraction.
mation of
dic device
Recommended