The 13th Pacific Symposium on Flow Visualization and Image Processing
7-10 August 2022, Tokyo, Japan

DEVELOPMENT OF VISUALIZATION TECHNIQUE FOR
HYDRODYNAMIC STRESS FIELD UTILIZING FLUORESCENT FORCE PROBE
R. KURIYAMA1, R. OKAMOTO2, W. YAMAMOTO1, H. KITAKADO3, S. SAITO3,K. TATSUMI1 and K. NAKABE1
1

Graduate School of Engineering, Kyoto University
Kyotodaigaku-katsura, Nishikyo-ku, Kyoto 615-8540 Japan
2

Faculty of Engineering, Kyoto University
Kyotodaigaku-katsura, Nishikyo-ku, Kyoto 615-8540 Japan
3

Graduate School of Science, Kyoto UniversityKitashirakawa Oiwake, Sakyo-ku, Kyoto 606-8502 JapanABSTRACTThis study investigates the fluorescence response of a molecular force probe called FLAP (flexibleand aromatic photofunctional systems) by measuring spatially averaged fluorescence spectra and twodimensional fluorescence images of FLAP solution in microchannel flow. We aim at developing atechnique for visualizing the hydrodynamic stress field through fluorescence imaging of FLAP. FLAP iscomposed of two rigid fluorescent aromatic wings fused with a flexible eight-membered ring. Thismolecule undergoes stress-induced conformational change between V-shape and planar shape and emits
blue or green fluorescence depending on its conformation. We used three types of FLAP whose molecular
ends were chemically functionalized with PEG (polyethylene glycol) chains of different molecularweights (5000, 10000, and 20000). Each FLAP-PEG was dissolved in a dimethyl sulfoxide (DMSO)/glycerol mixture, and the solution was supplied to a sudden contraction microchannel at constant flow
rates in the range of 2.2−446 µL/min. These flow rates correspond to the elongational stress of 0.1−21kPa at 10 µm upstream of the entrance of the contraction part. The spectral measurements showed thatthe intensity ratio of green (529 nm) to blue (494 nm) fluorescence increased monotonically with theflow rate, which indicates the response of FLAP to hydrodynamic elongational stress. In addition, FLAP
bearing the longest PEG chains showed the highest increasing rate of the intensity ratio, which suggests
the possibility of adjusting the stress sensitivity of FLAP-PEG by changing the chain length. In
fluorescence imaging, two-dimensional distributions of the intensity ratio were visualized around theentrance of the contractions part. The intensity ratio distributions showed a characteristic pattern in theregion where the velocity gradient was relatively large in the streamwise direction.
INTRODUCTION
From scientific and engineering perspectives, hydrodynamic stress is an essential quantity to analyze
the dynamic characteristics of flow fields. For example, wall shear stress is important in designing anddeveloping various fluidic systems since it is directly related to the flow resistance and required power for
fluid transport. Wall shear stress is also one of the key parameters in biomedical field which inhibits or
aggravates the pathogenesis of cardiovascular disorders(1). It is also important to know hydrodynamic stressin fluid as well as at channel wall, for understanding the unique behavior of non-Newtonian fluids andcomplex fluids. Therefore, various measurement techniques have been proposed to determine thehydrodynamic stress(2,3).
For the measurement of wall shear stress, a flush-mounted wall-element called floating-element is oftenused. This element is moveable in the plane of the wall-shear stress and its movement is detected byresistive or optical methods. Although a MEMS-fabricated floating-element has been developed recently,
the spatial resolution is still restricted to its characteristic size (~0.1 mm) and it is difficult to obtain two-The 13th Pacific Symposium on Flow Visualization and Image Processing
7-10 August 2022, Tokyo, Japandimensional distribution. In addition, installing the element might disturb the flow field. Other methods,
such as hot-wire anemometer, hot-film method, Preston and Stanton tubes, for instance, determine the wall
shear stress based on another measured quantity, i.e., velocity, heat transfer rate, or pressure. These methods
rely on the certain assumption concerning the flow field (such as law of the wall) and/or require extensive
calibration for accurate measurement. On the other hand, no well-established technique can be found forthe measurement of hydrodynamic stress in fluid. However, optical elasticity imaging utilizing
birefringence phenomenon in polymer solution has recently been studied as a promising approach(4).
For developing a non-invasive visualization technique for hydrodynamic stress field, the present study
proposes an optical-based method utilizing a unique molecular force probe called “FLAP (flexible andaromatic photofunctional systems)(5)”. In order to examine the feasibility of FLAP as a stress probe, weinvestigate the fluorescence response of FLAP to hydrodynamic elongational stress by measuring
spatially averaged fluorescence spectra and two-dimensional fluorescence images of FLAP solution in
microchannel flow.
PRINCIPLE
The molecular structure and the fluorescence characteristics of FLAP are depicted in Fig. 1. FLAP is a
fluorescent molecule which is composed of two rigid fluorescent aromatic wings fused with an eightmembered ring(5,6). FLAP changes its conformation flexibly between V-shape and planar shape and emits
blue or green fluorescence depending on its conformation. FLAP dissolved in solution takes the V-shape
conformation in the ground state and emits blue fluorescence after excited by ultraviolet (UV) light. If
FLAP is subjected to an external tensile force in the ground state, its conformation is altered from V-shape
to planar shape. FLAP in the planar shape is excited by the same UV light and emits green fluorescence.
Therefore, the intensity ratio of green to blue fluorescence can be used to quantify the force applied to themolecule. Since the force and the time required for the conformation change are on the order of 100 pN
and a few nanoseconds, respectively, FLAP can be potentially applied to real-time visualization of small
stress distributions in various fields. Based on this principle, FLAP has been used to visualize the tensile
force distribution during polymer deformation(7).
In the present study, we examine the fluorescence response of FLAP to hydrodynamic elongational
stress. We used three types of FLAP whose molecular ends were chemically functionalized with PEG(polyethylene glycol) chains with different molecular weights (5000, 10000, and 20000 for each end). The
total molecular weights (MW) of these molecules are 12000, 22000 and 42000. Hydrodynamic force isexpected to be exerted on the PEG chains, which are in coil state in stationary fluid, and stretch the FLAP
under flow condition. Since the critical elongational rate at which the polymer chain shows the initiation of
coil-stretch transition generally decreases as increasing the molecular weight of polymer(8), the differencein the molecular weights of PEG can change the stress sensitivity of FLAP functionalized with PEG chains(hereafter referred to as FLAP-PEG).
Force (~100 pN)

0Reversible change(~ ns)

1000 µm

50 µm

100 μmFlowBlue fluorescence
Green fluorescence

PDMSFig. 1. Reversible change in molecularconformation and fluorescence color of
FLAP in response to external force.

100 μmFig. 2. Schematic of a sudden contraction microchannel
and a transmission image of the measurement area offluorescence imaging.

The 13th Pacific Symposium on Flow Visualization and Image Processing
7-10 August 2022, Tokyo, Japan

EXPERIMENTAL SECTIONApparatus
The measurement system was mainly comprised of a microchannel, fluid supplying system, inverted
fluorescence microscope (Olympus, IX-73), and detector. Ultraviolet LED (Thorlabs, M365LP1) wasused as excitation light source. The fluorescence from the FLAP-PEG solution flowing in themicrochannel was collected by 100x objective lens (Olympus, UPlanSApo). Fluorescence mirror unit(Olympus, U-FUW) and multichannel spectrometer (Ocean Optics, Frame-S) were used for spectralmeasurement. On the other hand, fluorescence filters (Semrock, FF01-458/64 for blue and FF02-534/30
for green fluorescence) and CMOS camera (Andor, Zyla 4.2 plus) were used for fluorescence imaging.
A sudden contraction microchannel was used to evaluate the fluorescence characteristics of FLAP
probe in flow with elongational stress. Figure 2 shows the schematic of the microchannel composed of apolydimethylsiloxane (PDMS) chip and a cover glass. The width of the main channel was 1000 µm andthat of the contraction part was 50 µm. The channel height was 74.3 µm. A syringe pump (Nihon Koden,
CFV-3200) was used for supplying FLAP-PEG solution to the microchannel at constant flow rate.
Experimental condition
FLAP-PEG was dissolved into dimethyl sulfoxide (DMSO)/glycerol mixture (40:60 in volume) at theconcentration of 5 or 10 µM. The viscosity of the solution was 85.4 mPa·s at 296 K. The solution wassupplied to the microchannel at constant flow rates in the range of 2.2−446 µL/min. These flow rates
correspond to the cross-sectional average velocity of 10−2000 mm/s at the contraction part. The focal
plane of the objective lens was positioned at the middle height of the microchannel and the measurement
area was set at the entrance of the contraction part (Fig. 2). The exposure time of the spectrometer or thecamera was 1s and 30 spectra/images were collected for each condition.
Prior to the measurement, we conducted a numerical simulation of flow field using a computation
software (COMSOL Multiphysics 4.3a) and estimated the elongational stress along the center line of themicrochannel based on the velocity gradient in the flow direction. The elongational stress was estimatedto be 0.1−21 kPa at 10 µm upstream of the entrance of the contraction part for the above flow rate.

1.0

1.0

0.8

0.8
0.60.6480 494

529 540

10 mm/s
20 mm/s
50 mm/s
100 mm/s
200 mm/s
500 mm/s

0.40.20.0400

1.0Intensity ratio [-]Normalized intensity [-]RESULTS AND DISCUSSION
Spectral measurement
Figure 3 shows the fluorescence spectra of FLAP-PEG (MW=22000) solution at each flow rate, which
is normalized by the peak intensity at 494 nm. The legend shows the cross-sectional average velocity at
the contraction channel. The green fluorescence band (490−600 nm) increased with flow rate, whereasthat of blue fluorescence band (420−490 nm) decreased. This trend was also observed in the spectra ofFLAP-PEG with molecular weights of 12000 and 42000.
12000 (1st)12000 (2nd)22000 (1st)22000 (2nd)22000 (3rd)42000 (1st)

0.9

0.8

0.7

500

600

Wavelength [nm]700Fig. 3. Normalized fluorescence spectra of FLAPPEG (MW=22000) in DMSO/glycerol mixture ateach flow rate.

102

103

104

Elongational stress [Pa]Fig. 4. Relationship between the measured
fluorescence intensity ratio and theelongational stress estimated by simulation.

The 13th Pacific Symposium on Flow Visualization and Image Processing
7-10 August 2022, Tokyo, Japan

The intensity ratio of green (529 nm) to blue (494 nm) fluorescence of the measured spectra wasplotted against the elongational stress estimated by numerical simulation in Fig. 4. The legend represents
the molecular weight of FLAP-PEG and the measurement order in brackets. The intensity ratio increased
monotonically with the elongational stress for all types of FLAP-PEG. This indicates that FLAP wasplanarized by the flow stress and the number of FLAP in planar shape emitting green fluorescenceincreased with increasing the stress. This result suggests the potential application of FLAP as ahydrodynamic stress probe. In addition, FLAP bearing the longest PEG chains (MW=42000) showed thehighest increasing rate of the intensity ratio, which agrees with our expectations. However, the increasingrate of molecular weight 12000 was slightly larger than that of 22000. In addition, the number ofmeasurements is still limited since the measurements were sometimes disturbed by channel clogging andleakage. Further measurement should be made to ensure the measurement reproducibility and verify therange of the stress sensitivity of FLAP-PEG by changing the PEG chain length.
Two-dimensional fluorescence imaging
To examine the possibility of flow stress visualization using FLAP-PEG, two-dimensional
fluorescence imaging was performed at the measurement area shown in Fig. 2. FLAP-PEG withmolecular weight of 22000 was used for the experiment. The flow rate was adjusted in the range 2.2−111µL/min so that the cross-sectional average velocity was 10−500 mm/s in the contraction channel.
Fluorescence images of blue and green bands were measured by switching the fluorescence filters
corresponding to these spectral bands after the flow reached the steady state at each condition.
Figures 5(a)−(c) show the distributions of the green to blue fluorescence intensity ratio calculated fromthe measured images at the condition of 10, 50 and 100 mm/s, respectively. The white area in the figure
represents the channel wall. The intensity ratio increased in all area with increasing the flow rate. The
intensity ratios averaged over the area approximately 10 µm upstream of the entrance (indicated by red
square in Fig. 5(a)) are 0.64, 0.71, 0.76 and 0.82 at the condition of 10, 50 100, and 500 mm/s, whichcorresponds to the elongational stress of 0.1, 0.5, 1.0, and 5.2 kPa, respectively. The intensity ratio
increases almost linearly with the elongational stress in logarithmic scale. This is consistent with thespectral measurement result in Fig. 4. Focusing on the distribution, all figures exhibit a similar fan-likepattern, in which the area of high intensity ratio is expanding from the entrance of the contraction to theupstream area. Since large velocity gradient is generated in the streamwise direction upstream of theentrance, it is reasonable that the intensity ratio is high in the region. However, area of heigh intensity
ratio can be observed also downstream of the entrance (i.e., in the contraction part). The velocity gradientin the flow direction decreases rapidly in the region and the fluorescence intensity ratio should decrease(a) 10 mm/s(b) 50 mm/s

0(c) 100 mm/s

Intensity ratio [-]0.9y [µm]200.8400.7600.680
1000204060x [µm]80100 0204060x [µm]80100 0204060x [µm]801000.5Fig. 5. Distributions of the intensity ratio of green (529 nm) to blue (494 nm) fluorescence measured
at different flow rates. The cross-sectional average velocity in the contraction channel was (a) 10 mm/s,(b) 50 mm/s, and (c) 100 mm/s.

The 13th Pacific Symposium on Flow Visualization and Image Processing
7-10 August 2022, Tokyo, Japan

as well. In addition, some influence of the intensity profile of the excitation light is observed in the results
as can be seen in the lower left region of the figures. We will further analyze the intensity ratiodistributions in flow fields with various channel geometries and compare with numerical simulation
result to examine if the intensity ratio distribution has some correlation with stress distribution.
CONCLUSIONSThe present study investigated the change of fluorescence wavelength of FLAP-PEG in response tohydrodynamic elongational stress in order to develop a non-invasive visualization technique for stress
distribution in channel flow. The spectral measurement result in the sudden contraction microchannel
showed that the intensity ratio of green to blue fluorescence monotonically increased with theelongational stress and the highest increasing rate was observed for FLAP-PEG with the highest
molecular weight. The two-dimensional distributions of the intensity ratio exhibited a characteristic
pattern around the entrance of the contraction part where the relatively large velocity gradient wasgenerated in the flow direction. In addition, the intensity ratio in the whole measurement area increased
with the elongational stress. These results indicate the response of FLAP-PEG to hydrodynamic stressand support the potential use of the molecule for stress visualization in flow field.
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