Proceedings of the 4th International Forum on Heat Transfer, IFHT2016
November 2-4, 2016, Sendai, Japan

IFHT2016-1987

FLUID TEMPERATURE MEASUREMENT IN MICROCHANNELSUSING FLUORESCENCE POLARIZATION METHOD
Atsushi Suzuki*, Chi-Hsuan Hsu, Kazuya Tatsumi, Reiko Kuriyama, Kazuyoshi Nakabe
Kyoto University, Kyotodaigakukatsura, Nishikyo-ku, Kyoto 615-8540, Japan

ABSTRACT
A fluid temperature measurement technique based on fluorescence polarization is developed and applied tomeasure the two-dimensional temperature distributions in microchannel. The fluorescence depolarization
due to the Brownian rotational motion of the fluorescent molecules is measured in this method and isconverted to the fluid temperature. Since the fluorescence polarization degree is independent of fluorescenceintensity, the fluorescence polarization is less affected by the quenching effects, which is one of thedrawbacks of LIF (laser-induced fluorescence). Experiments were performed using a microchannel andfluorescent molecules solved in liquid. In order to examine the validity of the measurement, the effects of thefluid viscosity and temperature on the fluorescence polarization were measured. Further, linear temperature
gradient was generated in the microchannel and measured by the present method. The results showed that as
the fluid viscosity decreased and the temperature increased, the fluorescence depolarization increased. The
results agreed well with the theoretical values and thermocouple measurements confirming the validity andthe feasibility of the method.
KEY WORDS: Fluorescence polarization, Fluid temperature measurement, Microscopy, Microchannel, FITC

1. INTRODUCTION
Microscale temperature measurement is an important issue in micro-devices so called lab-on-a-chip andmicro total analysis system (μ-TAS). For example, in biochemical reaction represented by the DNA
hybridization process requires a temperature control and, therefore, measurement of an accuracy of ±1°C [1].
The conventional techniques in measuring the fluid temperature in the microchannel are thermocouple [2],resistance thermometer [3], and laser-induced fluorescence (LIF) [4] methods. Thermocouples and resistancethermometers are relatively reliable, compact and low in cost. However, since these probes are intrusive andhave a characteristic size relative larger than or equal to the microchannel, the spatial resolution is low. LIF,
which is based on the temperature dependence of the fluorescence intensity, overcomes this limitation andallows us to obtain the fluid temperature with high spatial resolution. However, there are some issues in themeasurement using LIF: the fluorescence intensity is affected by the variation of the excitation light intensityand quenching effects related to factors other than temperature.
To tackle these problems, a novel method measuring fluorescence polarization is proposed in this study. The
depolarization degree of the fluorescence emitted from the fluorescent molecules, which are exposed tolinearly polarized light, can be an index to measure the fluid temperature. In the present study, therelationship between the polarization degree and fluid temperature is measured for microchannel flow inorder to validate and examine the feasibility of the measurement method.

*Corresponding Author: tatsumi@me.kyoto-u.ac.jp

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IFHT2016-1987

Fig. 1 Schematic of the relationship between the polarized excitation light and the polarization degree ofthe fluorescence in the cases of the molecules in the stationary state and the presence of Brownian motion.
The probability density distribution shows the probability of the directions of the absorption andfluorescence moments.

2. PRINCIPLE
The physics of the temperature measurement method using the polarization degree of fluorescence isdescribed in this section. Figure 1 shows the relationship between the polarization directions of the linearlypolarized excitation light, absorption moment of the fluorescent molecule and fluorescence. Among themolecules exposed to the excitation light, those with the absorption moment having a component aligned
parallel to the polarization direction of the excitation light are excited. The absorption degree of thesemolecules depends on the angle between these directions, θ, and can be expressed by the probability density
function of absorption which is proportional to cos2θ. The excited fluorescent molecules will then emit
fluorescence polarized in the same direction with the absorption moment.
In the case of fluorescent molecules in stationary state without rotation, the fluorescence is polarized in thesame direction with the excitation light with some variation owing to the probability density function
mentioned previously. On the other hand, when the fluorescent molecules are dissolved in fluid, therotational motion due to Brownian motion influences the polarization direction of the fluorescence. In thiscase, the excited molecules experience the rotational motion during the period of the excitation and emission,and the polarization direction of the fluorescence shows greater randomness compared with the fluorescenceof molecules in stationary condition. Namely, depolarization is observed.
To consider the relationship between the depolarization degree and other parameters, a variable called thepolarization degree P is first defined as follows.
𝑃=𝐼∥ − 𝐼⊥𝐼∥ + 𝐼⊥(1)𝐼∥ and 𝐼⊥ present the fluorescence intensities of the components that are parallel and perpendicular to thepolarization direction of the excitation light, respectively. Perrin [5] and Weber [6] have theoretically derived
Eq. (2) to express the characteristic of P of fluorescent molecules with Brownian motion.
1 1
1 1
𝑘B 𝑇
( − ) = ( − ) (1 +𝜏)
𝑃 3
𝑃0 3𝜇𝑉(2)P0 is the polarization degree of the molecule in steady state (without rotation). kB is the Boltzmann constant.
μ and T are the viscosity and temperature of the fluid, respectively. τ and V are the fluorescence relaxation
time and volume of the molecule, respectively. As one can see in Eq. (2), the reciprocal number of P shows alinear relation with T/μ. Therefore, if the molecular volume remains constant during the measurement, thefluid temperature can be obtained by measuring the polarization degree of the fluorescence. The presentmeasurement is based on this physics.

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3. MEASUREMENT SYSTEM
Measurement was conducted by upright microscope (Olympus, BX51) and LED (Thorlabs, M490L3) lightsource. Figure 2(a) shows the schematic of optical system. To separate the excitation light and fluorescence, thefluorescence excitation filter (Semrock, FF01-482/35-25), dichroic mirror (Semrock, FF506-Di03-25x36), thefluorescence filter (Semrock, FF01-536/40-25) were placed in the optical path. Fluid viscosity effects on
fluorescence polarization were measured by transmission observation. The excitation light from the LED waslinearly polarized by the polarizing light condenser (Olympus, U-POC-2) and illuminated the fluid in themicrochannel. The fluorescence through analyzer (Olympus, U-AN360P-2) was measured by photomultiplier(Hamamatsu Photonics, H5783-03). On the other hand, fluid temperature effects on the fluorescencepolarization were measured by epi observation. The excitation light passed through polarizer (Olympus, U-PO3),
and the fluorescence observed through rotatable polarizer (Olympus, LP40120) was received by high sensitivity
camera (Andor, Zyla DV435) with an exposure time of 1s.
Figure 2(b) shows the microchannel fabricated by PDMS and the temperature control stage. The channel wasa straight one with the height of 43μm and width of 850μm. The stage was made of aluminum and composedof a bottom plate and two top plates. The top plates were set parallel with 1mm spacing. Between the bottom
plate and each top plate, two peltier devices were placed to control the stage temperature. Although notshown here, the plate temperature was measured by thermography (FLIR systems, ThermoCAM E65) andthermocouples when the stage was heated, and it was validated that the surface temperature of the stage wasuniform. The microchannel was placed on the top plates with the channel direction set perpendicular to thegap of the plates. Three thermocouples were attached to the backside of the channel bottom wall (coverglass) equally spaced by approximately 300μm.

(a) Optical path of measurement(b) Microchannel and temperature control stage
Fig. 2 Schematics of the (a) optical path in the microscope and (b) straight microchannel andtemperature control stage used for temperature measurement.

4. RESULTS AND DISCUSSION
4.1 The Viscosity Effects on Fluorescence Polarization
Fluid viscosity effects on the fluorescence polarization are discussed in this section to compare with thetheoretical value for validation and to discuss the effects of the fluorescent probe used in the measurement.
To change the fluid viscosity, glycerol (Nacalai Tesque, #17018-25) was mixed with water. Two fluorescentmolecules, which have different volume, were examined. One is fluorescein isothiocyanate (FITC,MW=389: Nakalai Tesque, #V9E9989) and the other is Casein-FITC conjugated (C-FITC, MW=26000:

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AAT Bioquest, #13440). FITC and C-FITC have different molecular weight. Their size are approximately
1nm and 10nm, respectively.
Figure 3 shows the relationship between the inverse value of fluid viscosity, 1/μ and fluorescencepolarization degree, P. The theoretical values represented by the solid line are derived from Eq. (2) under thecondition shown in Table 1. In the case of FITC, P decreases as the fluid viscosity decreases. The Brownian
motion becomes more vigorous as μ decreases and depolarization is observed. Comparing the experimental
results with the theoretical ones, the two values agree reasonably well showing the validation of the presentmeasurement.
In the case of C-FITC, P or the theoretical value is constant against μ. This shows that no rotational motion
exists during the period of the excitation and emission. However, the experimental values decrease largely inthe region approximately at 1/μ = 50 Pa-1∙s-1. Although not shown here, the particle diameter was measured
based on dynamic light scattering method (Otsuka electronics, ELSZ-2plus). The results showed that thediameter largely decreases at approximately 1/μ = 50 Pa-1∙s-1. This result suggests the possibility that theFITC (or a part of C-FITC) detached from the casein at this condition. Since the isolated molecular or clusteris smaller than C-FITC, P is decreased in the region at approximately 1/μ > 50 Pa-1∙s-1.
Under the fluid viscosity condition of water, 1/μ ≅ 1000-1200 Pa-1∙s-1, P of C-FITC case shows greater value
compared with that of FITC case. This shows a larger signal in the measurement. Therefore, C-FITC wasused for the temperature measurement to increase the S/N ratio.
0.6C-FITC

0.5
FITC water solution
C-FITC water solution
Theory (Eq. (2))

P

0.4
0.3
0.2
0.1
0.0

FITC

0200

4006008001000 1200

1/ [1/Pas]

Fig. 3 Fluid viscosity μ effects on the fluorescence polarization P in the cases of FITC and C-FITC
solutions. The solid lines present the theoretical values calculated by Eq. (2) applying the values shown in
Table 1.
Table 1 Properties applied to Eq. (2) to calculate P of FITC and C-FITC shown in Fig. 3.
Properties
FITCC-FITC
P0
0.5
0.5
kB
1.38 × 10-23 J/K
1.38 × 10-23 J/K
T
303 K303 Kτ
4 ns4 nsd
1.08 nm
10 nm

4.2 Fluid Temperature Measurement
Calibration. Calibration was performed on the fluorescent polarization degree, P, of C-FITC 0.1wt% aqueous
solution for fluid temperature in the temperature range from 28 to 38°C. This is a reasonable range considering
the temperature applied to the biochemical assay. The temperature in the channel was kept constant at each4IFHT2016-1987
temperature condition. The fluorescence intensity and P distributions in the area of 416μm × 770μm shown inFig. 2(b) was measured by the camera.
The spatial systematic error of the optical systems, B(x, y), was calculated using the following formula and was
subtracted from each image.

B( x, y ) 1 N
 ( Pi ( x, y)  Pi )N i 1(3)

Pi is a spatially averaged value of the ith image and N is the number of images.

Figure 4 shows the results of the calibration. The plots in the graph are (P – B) averaged over the area and of tenimages. The straight line drawn in the figure was derived on the basis of the least square mean approximation.
The error bars were calculated from ten images and they present the 95% confidence range for theuncertainty of each temperature condition. In the figure, 1/P increases linearly against the fluid temperature Twith the relation presented as Eq. (4).
1− 6.43
𝑃
𝑇=0.324(4)

The accuracy of the present method was ±2.99°C. This empirical relation matches to Eq. (2) and confirms thevalidity of the present method.
201/P181614283032343638

T [oC]

Fig. 4 Relationship between fluid temperature and reciprocal of P of C-FITC solution. The solid line isbased on the least mean square approximation.

42T [oC]

550

40
39
38
37
36
35
3433323130
2928

y [m]

500
450
400
350
300
250
200

300

400

500

600

700

800

38
3634
3230
28
26

900

x [m]

Thermocouples
The fluorescence polarization method

40

T [oC]

600

0200

400

600

800

1000

x [m](a) Two-dimensional distribution(b) Temperature in the x direction
Fig. 5 The measured temperature distribution in microchannel: (a) Two-dimensional distribution, (b) Averaged
temperature in the region with the size of 320μm × 100μm dividing the overall area into six segments.

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Temperature measurement. Temperature measurement was then performed in the case when a temperature
gradient was generated in the microchannel to validate the local temperature measurement. The temperaturesof the left and right of the thermocouples among the three ones shown in Fig. 2(b) were controlled to be36°C and 28°C, respectively. The distance between these thermocouples was 743μm and temperature differenceis 8°C. Therefore, linear temperature gradient of -10.6°C /mm was generated in the channel.
Figure 5(a) shows the two-dimensional temperature distribution in microchannel measured by the presentmethod. As the x value increases, the measured temperature decreases. The result qualitatively agrees withthe temperature gradient generated by the stage. In order to evaluate this temperature gradient quantitatively,
temperature in the six regions were averaged. These regions had the area of 320μm × 100μm and werelocated to be equally spaced in the flow direction (x axis). The results are shown in Figure 5(b) by the squaresymbols. The black circles shown in the graph present the temperature measured by the thermocouples attachedto the backside of the channel bottom wall. The measured value using the fluorescence polarization method
decreases linearly with x. Comparing the results obtained by P and thermocouples, both slopes agree well witheach other: the temperature gradient of both measurements are -10.3°C /mm and -10.6°C /mm, respectively.
Fig. 5(b) also shows that the absolute values measured by the present method were little larger than thethermocouple measurements. There is a possibility that the positions of the thermocouples were slightly shiftedto the right. Due to the thermocouple thickness, it was difficult to attach the thermocouples in the accuracy of
micro meter scale.

5. CONCLUSIONS
Experiment was performed to study the feasibility of the fluid temperature measurement using thefluorescence polarization method. The fluid viscosity effects on P showed a negative correlation and agreed
well with theoretical values. Using Casein-FITC conjugated molecule solution, the reciprocal value of P
showed a linear relationship with the fluid temperature and accuracy of ±2.99°C. Further, the temperature
distribution in the microchannel was measured and compared with the values measured by thermocouples.
The results agreed well and confirmed the feasibility of the present method.

NOMENCLATURE
Bd𝐼∥ , 𝐼⊥
kBP, P0

systematic error(-)
diameter( nm )
fluorescence intensity(-)
Boltzman constant(J/K)
fluorescence polarization degree ( - )TVθμτtemperaturevolumeangle
viscosity
fluorescent life time(K)( m3 )( rad )
(Pa∙ s)(s)

REFERENCES
[1]
[2]
[3]
[4]

[5]
[6]

Kim, Y.H., Yang, I., Bae, Y.S. and Park, S.R. “Performance evaluation of thermal cyclers for PCR in a rapid cycling condition,”
Bio Techniques, 44, pp.495-505, (2008). Journal Paper
Debey, D., Bluhm, R., Habets, N. and Kurz, H. “Fabrication of planar thermocouples for real-time measurements oftemperature profiles in polymer melts,” Sensors and Actuators A, 58, pp.179-184, (1997). Journal Paper
Glasser, H., Schnelle, T., Mü ller, T. and Fuhr, G. “Electric field calibration in micro-electrode chambers by temperature
measurements,” Thermochimica Acta, 333, pp.183-190, (1999). Journal Paper
Yoon, S.Y. and Kim, K.C. “Signal intensity enhancement of μ-LIF by using ultra-thin laser illumination and aqueous mixture
with ethanol/methanol for micro-channel applictions,” Optics and Lasersin Engineering, 44, pp.224-239, (2006). Journal
PaperPerrin, F. Ann. Phys. (Paris), 12, 169, (1929). Journal PaperWeber, G. Adv. Protein Chem., 8, 415, (1953). Journal Paper

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