Title

Micro-scale temperature measurement method using
fluorescence polarization

Author(s)Tatsumi, K.; Hsu, C. H.; Suzuki, A.; Nakabe, K.

Citation

Journal of Physics: Conference Series (2016), 745

Issue Date

2016-09

URL

http://hdl.handle.net/2433/218803

Right© Published under licence by IOP Publishing Ltd. Content
from this work may be used under the terms of the Creative
Commons Attribution 3.0 licence. Any further distribution ofthis work must maintain attribution to the author(s) and the titleof the work, journal citation and DOI. Published under licence
by IOP Publishing Ltd 1

Type

Conference Paper

Textversion

publisher

Kyoto University

Home

SearchCollections

Journals

About

Contact us

My IOPscience

Micro-scale temperature measurement method using fluorescence polarization

This content has been downloaded from IOPscience. Please scroll down to see the full text.
2016 J. Phys.: Conf. Ser. 745 032097(http://iopscience.iop.org/1742-6596/745/3/032097)
View the table of contents for this issue, or go to the journal homepage for more
Download details:
IP Address: 130.54.110.33
This content was downloaded on 13/03/2017 at 05:31
Please note that terms and conditions apply.

You may also be interested in:
Numerical calculation of dielectrophoretic and electrostatic forces acting on micro-scale particles
M Praeger, Z Li, J M Smallwood et al.
Sub-millimeter arbitrary arrangements of monolithically micro-scale electrical double layer
capacitors
Karolina U Laszczyk, Kobashi Kazufumi, Shunsuke Sakurai et al.
Analytic Solution for Steady Slip Flow between Parallel Plates with Micro-Scale Spacing
Zhang Tian-Tian, Jia Li and Wang Zhi-Cheng
Numerical study of micro-scale gas flow using finite volume method
A Chaudhuri, C Guha and T K Dutta
Multi-physics analysis for MEMS meshing micro-gear contacts
S Theodossiades, M Teodorescu and H Rahnejat
Geometry generation challenges for modelling and analysis of micro-structured materials
A J McMillan
Resolution of structure characteristics of passive acoustic emission signals in multiphase flow
system
Yi-Jun He, Jing-Dai Wang and Yong-Rong Yang
Fabrication of Stainless Steel Mold Using Electrochemical Fabrication Method for Microfluidic
Biochip
Min-Soo Cho, Hyun-Woo Lim, Caroline Sunyong Lee et al.

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

Micro-scale temperature measurement method using
fluorescence polarization
K. Tatsumi, C-H. Hsu, A. Suzuki and K. Nakabe
Department of Mechanical Engineering and Science, Kyoto University,
Kyotodaigakukatsura, Nishikyo-ku, Kyoto, Kyoto 6158540, JAPANE-mail. tatsumi@me.kyoto-u.ac.jp
Abstract. A novel method that can measure the fluid temperature in microscopic scale bymeasuring the fluorescence polarization is described in this paper. The measurement technique
is not influenced by the quenching effects which appears in conventional LIF methods and isbelieved to show a higher reliability in temperature measurements. Experiment was performed
using a microchannel flow and fluorescent molecule probes, and the effects of the fluid
temperature, fluid viscosity, measurement time, and pH of the solution on the measured
fluorescence polarization degree are discussed to understand the basic characteristics of thepresent method. The results showed that fluorescence polarization is considerably less sensible
to these quenching factors. A good correlation with the fluid temperature, on the other hand, wasobtained and agreed well with the theoretical values confirming the feasibility of the method.

1. Introduction
Microfluidic device are considered to be powerful tools in the fields of chemistry, biology and medicine
in terms of enhancing the accuracy, sensitivity, and throughput and reducing the cost and time of thediagnoses and analysis. They are called lab-on-a-chip and micro total analysis system (TAS). In orderto develop and design these devices, measurement methods that can measure the fluid temperature in
microchannels is an important issue to be solved. For example, precise temperature management withaccuracy of 0.1C is generally required to control the chemical and biological reaction occurring in the
microchannel, represented by the process in the DNA hybridization [1].
The conventional methods employed to measure the fluid temperature in microchannels are thethermocouples, resistance temperature probe and laser induced fluorescence (LIF) method. Among these,thermocouples and resistance sensors are relatively reliable [2]. However, the spatial resolution is low(>10m), and the location of the temperature measurement is fixed and the number of points are limited.
The fabrication process of the wire patterning on the microchip, which is complicated and time
consuming, is another problem. LIF, which is based on fluorescence intensity measurement of thefluorescent molecules solve in the fluid, is therefore often used since it can perform a noninvasive andtwo-dimensional measurement [3]. Nevertheless, there are issues which affects the accuracy of themeasurement using LIF. One is the variation of the light intensity due to the light source and pathways.
The other is the quenching effect.
Quenching effects [4][5][6] is a phenomena which decreases the fluorescence intensity and resultsfrom several reasons: temperature, oxygen, concentration and photobleaching. Temperature quenchingis a type of excited-state reactions caused by encounters related to molecular rotational motion and the
motions of quencher substance. Due to this quenching effect, the fluorescence intensity decreases with
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

temperature and the LIF measurement is based on this relationship. Oxygen plays an important role asquencher in the quenching process due to its high solubility in solutions. The collision between
fluorescent molecules and oxygen forms an unilluminated substance and decreases the fluorescence.
Therefore, fluorescence intensity decreases with oxygen concentration.
If the concentration of the fluorescent molecules are relatively high, concentration quenching or inother words, self-quenching takes place. This phenomenon is due to resonant transfer of energy from an
excited molecule to an unexcited one, a transfer that makes spatial delocalization of the excitation. The
increase of the absorption rate due to the high concentration will result in the elimination of excitation
light and decreases of the fluorescence. Photobleaching is another significant phenomenon in terms of
the fact that fluorescent molecules lose the ability of fluorescence emission when exposed to a strong
excitation light. This results from the chemical damage of the molecular structure and is generally
irreversible. The quenching effects mentioned here will affect the relation between the temperature andfluorescence intensity and deteriorate the performance of LIF method.
To tackle these problems, a novel fluid temperature measurement method based on the fluorescencepolarization measurement is proposed in this study. As will be discussed in detail in Section 2, when themolecule is exposed to a polarized excitation light, a polarized fluorescence is emitted from thefluorescent molecule. Depolarization, a decrease of the polarization degree, takes place when themolecule experience the Brownian rotational motion in fluid. The degree of this depolarization depends
mainly on the fluid viscosity, temperature and volume size of the molecule. Therefore, if the molecule
size is fixed, the polarization degree of the fluorescence will depend on the fluid temperature and canbe an index to measure the fluid temperature.
In the present study, the relation of the polarization degree of the fluorescent and fluid temperatureis measured for microchannel flow. In addition to this, the relation of the fluid viscosity and polarization
degree P is measured and compared with the theoretical values to validate the present method. Further,
the effects of the fluid pH on the fluorescence polarization and intensity are measured under stationary
fluid case to evaluate the measurement performance in the presence of quenching effect.

2. Measurement physics
The present method measures the polarization degree of the fluorescence emitted from the molecules.
The fundamental principles of the measurement method will be described in this section.
Figure 1 shows the relationship between the polarization directions of the excitation light,
fluorescence, and absorption moment of the fluorescent molecule. As shown in Fig. 1(a), when the
fluorescent molecules are exposed to an excitation light linearly polarized (z axis in Fig. 1 (a)), thefluorescent molecules of which the absorption moment direction is parallel to the z axis are excited. The
absorption degree of the molecule to the excitation light depends on the angle between the absorption
moment and the polarization direction of the excitation light, . As  increases, this absorption rate
decreases in the rate of cos2. Therefore, if the direction of the absorption moment is parallel to thepolarization direction of the excitation light (z direction), the absorption rate of the molecule becomesmaximum. On the other hand, the molecule will not be excited if the direction of the absorption
momentum is perpendicular to the excitation light (x and y directions).
The emitted fluorescence is also polarized in the same direction with the absorption moment. In otherwords, if an infinite number of fluorescent molecules randomly positioned are exposed to a linearlypolarized excitation light, the molecules with the components of the absorption moment parallel to theexcitation light are stochastically excited. The fluorescence observed from these molecules is, therefore,
mainly polarized in the same direction with the excitation light.
When the molecules are suspended in the fluid, the rotation of the molecule due to the Brownianrotation significantly influences the polarization direction of the fluorescence in addition to thestochastic effects of the absorption moment. That is, since the molecule rotates during the period of thefluorescence excitation and emission, the polarization direction of the fluorescence shows larger2

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

Figure 1. Schematic of the relationship between the polarized excitation light and thepolarization degree of the fluorescence in the cases of the molecules in stationary state and thepresence of Brownian motion. The probability density distributions show the probability of thedirections of the absorption and fluorescence moments.
variance with the direction of the excitation light compared with those of fluorescent molecule instationary condition (See Fig. 2(b)). This is the depolarization of the fluorescence.
To consider the relationship between the depolarization degree and other parameters, a variable
called the polarization 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 tothe polarization direction of the excitation light, respectively. Perrin [7] and Weber [8] have theoreticallyderived Eq. (2) to express the characteristic of P of a fluorescent molecule with Brownian motion.
k T 
 1 1   1 1       1  B  
P
3
P
3
V 

  0


(2)
P0 is the polarization degree of the molecule in steady state (without rotation). kB is the Boltzmann
constant.  and T are the viscosity and temperature of the fluid, respectively. andV are the fluorescencerelaxation time and the volume of the molecule, respectively. As one can see in Eq. (2), the reciprocal
number of P shows a linear relation with the T/. Therefore, if the molecular volume remains constantduring the measurement, the fluid temperature can be obtained by measuring the polarization degree ofthe fluorescence. The present measurement is based on this physics.

3

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

The total fluorescence intensity I can be influenced by various factors. For example, I can bepresented by Eq. (3).


I = I0c

I0 is the intensity of the excitation light and c is the concentration of the florescent molecules.  isthe quantum efficiency, and  is the absorption coefficient presenting the rate that the excitation light isabsorbed while it passes through a unit length of solution.
As mentioned in Section 1, the quenching effect occurs attributed to various reasons. The change ofthe fluid temperature also produces a change in the vibration motion of the molecule, hence, thefluorescence intensity. Fluid temperature measurement using the laser induced fluorescence (LIF)
method employs this relationship between the fluid temperature and fluorescence intensity. On the other
hand, this means that the measurement performance can be influenced by other quenching effects andthe disturbance of the light intensity at the light source and optical path.
The present method measures the polarization degree, P. As shown in Eq. (1), P is a value normalized
by the total fluorescence intensity I//+I. P is, therefore, not influenced by the variation of thefluorescence intensity. This indicates that the previously mentioned quenching effects can be neglectedin this method.

(a) Optical path of measurement(b) reservoir (stationary fluid)(c) microchannel

Figure 2. Schematics of the (a) optical path in the microscope, (b) cylindrical reservoir withstationary fluid used to measure the pH, and (c) straight microchannel used to measure the fluid
viscosity and temperature effects.
3. Measurement method
Measurement was conducted using an uplight optical microscope (Olympus, BX51), LED (Thorlabs,
M490L3) as the light source, and photomultiplier (Hamamatsu Photonics, H5783-03) as the detector.
Figure 2(a) shows the schematic of the optical pathway. The light from the LED passes the condenser
with polarizer (Olympus, U-POC-2), fluorescent excitation filter (Semrock, FF01-482/35-25) andobjective lens (Olympus, LMPlanFLN) and illuminates the fluid in the microchannel. The fluorescenceis measured through the fluorescence filter (Semrock, FF01-536/40-25) and analyzer (Olympus, UAN360P-2).
Measurement of the fluid pH effects on polarization was carried out under the stationary fluid
condition using a PDMS reservoir. Measurements of the fluid viscosity and temperature effects on thepolarization were performed using the microchannel flow. Figure 2(b) shows the schematic of the47th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

reservoir. The reservoir was made of PDMS(poly-dimethylsiloxane) with cover glasses sealing the top
and bottom sides. The diameter and height of the reservoir are 5mm and 3mm, respectively
Figure 2(c) shows the schematic of the microchannel. The channel is a straight one with the heightof 43m and width of 850m. The microchannel was fabricated by PDMS using the standard
photolithography and soft lithography. The channel pattern was first printed on the photomask using thelaser lithography system. The pattern was then transferred to SU-8 (MicroChem Inc.) spin coated on
silicon wafers using photoresists and lithography systems, and was applied as the mold in the soft
lithograph. PDMS was first degassing by decompression and then casted using the SU-8 mold. Holes
were cored at the inlet and outlet of the channel with biopsy puncher. The PDMS was then attached tothe cover glass. To control the fluid temperature, the microchannel was placed on the temperature
controlled stage. Thermocouples were attached to the cover glass at the locations 1mm aside of thechannel and are referred as channel temperature.
Two fluorescent molecules are employed in the measurement. One is the fluorescein isothiocyanate(FITC: Nacalai Tesque, #V9E9889) and the other is casein-FITC conjugated (C-FITC: AAT Bioquest,#13440). C-FITC is a casein which is labeled by several FITCs. The molecular weight MW of FITC and
C-FITC are different and are MW=389 and 26000, respectively. Therefore, the size of the molecules areapproximately 1nm and 10nm, respectively. As shown in Eq. (2), the polarization degree P of thefluorescence emitted from the molecule relies on the temperature, the fluid viscosity and the volume of
the molecule. Larger volume implies larger P leading to increase of the S/N ratio of the measurement.
Further, the gradient of P against temperature T increases under larger P conditions showing that ahigher sensitivity and accuracy of temperature measurement can be obtained with larger P. Therefore,
C-FITC with larger MW (volume) compared with FITC was examined in this study.

I/I7.1, P/P7.1

11
10

I/I7.1 of FITC

9

P/P7.1 of FITC

8

I/I7.1 of C-FITC

7

P/P7.1 of C-FITC

6
5
4
3
2
1
0
6.5

7.0

7.5

8.0

8.5

9.0

9.5 10.0 10.5 11.0

pH

Figure 3. pH effects on fluorescence intensity I and polarization degree P in the cases of
FITC and C-FITC solutions. I and P are normalized by the values of pH=7.1.
4. Results and discussion
4.1. Fluid pH effects
Fluorescent molecules are strongly pH dependent [9] and the effect of the fluid pH on the fluorescentintensity and polarization are evaluated in this study. The pH of the solution was controlled my adding
NaOH to the solution following the next procedure. A solution of 0.75 mol/L NaOH was preparedinitially and added to Millipore ultrapure water to obtain the target pH value. The sample solutions ofcasein-FITC with different pH were prepared at a concentration of 0.01wt% using the pH controlled

57th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

ultrapure water as solvent. For the FITC solution, 0.001wt% FITC of the same aqueous solution was
prepared.
Figure 3 shows the effects of the pH on the fluorescence intensity I=I∥ +2I⊥ and the fluorescencepolarization P. The results are normalized by the values of pH=7.1, respectively. I/I7.1 takes the
maximum peak approximately at pH=10, which agrees well with the results of Obare [9]. The maximum
peak shows 3 and 10 times larger values than those of pH=7.1 in the cases of FITC and C-FITC,
respectively. This indicates that quenching by the pH will significantly affect the measurement accuracy.
On the other hand, the variation of P/P7.1 in the cases of FITC and C-FITC are both less than 20%.
These values are much smaller than those of I/I7.1. This shows that the fluorescence polarization is lessaffected by the pH conditions and high reliability and accuracy of temperature measurement can be
performed applying the measurement.
4.2. Fluid viscosity effects
Fluid viscosity effects on the fluorescence polarization is discussed in this section to compare with thetheoretical value and evaluate the effective fluorescent molecule for the measurement. To control theviscosity of the fluid glycerol (Nacalai Tesque, #17018-25) was mixed with the solution.
0.6
C-FITC
0.5
FITC water solution
C-FITC water solution
Theory (Eq. (2))

P

0.4
0.3
0.2
0.1

FITC

0.0
0

200

400

600

800

1000

1200

1/ [1/Pa s]

Figure 4. Fluid viscosity  effects on P in the cases of FITC and C-FITC solutions. The solidlines presents the theoretical values obtained by Eq. (2) applying the values shown in Table 2.
Table 1. Properties applied to Eq. (2) to calculate P of FITC and C-FITC.
properties

FITC

C-FITC (casein-FITC conjugated)

P0
T

0.5
1.38×10-23 J/K
303K

0.5
1.38×10-23 J/K
303K



4ns

4ns

d

1.08 nm

10 nm

B

Figure 4 shows the relation of P and the fluid viscosity  in the case of FITC dissolved in water. The
theoretical values derived from Eq. (2) are shown by the solid line in the figure for comparison. This6

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

line is calculated applying the condition shown in Table 1, in which the fluorescence lifetime anddiameter, τ and dFITC, in the case of FITC referred to Magde [10] and Fowlkes [11] are shown.
In Fig. 4, the polarization degree P of the FITC decreases as the solution viscosity decreases. As thefluid viscosity decreases, the Brownian motion of fluorescent molecules become more active and the
direction of the absorption moment vector become random. This leads to larger depolarization. In thiscase, 1/P and 1/ show a linear relation as presented in Eq. (2). Comparing the experimental results withthe theoretical ones, the two values agree reasonably well showing the validity of the present
measurement apparatus.
Together with FITC case, the results of fluorescence polarization in the case of casein-FITC areshown in Fig. 4. Same as the FITC case, the solid lines present the theoretical values obtained from Eq.
(2) and Table 1, in which the diameter refers to Dalgleish [12]. It should be noted that several
assumptions were made to apply this equation to the casein-FITC-conjugated molecules. One is that the
FITC molecules are attached to the casein with random position and orientation. The other is that the
FITC molecules are fixed to the casein and will not move and change its relative direction to the casein.
In this case, the fluorescence polarization is related to the rotation of casein and is solved by Eq. (2).
Compared with the FITC case, the theoretical value shows that the depolarization of the casein-FITC
due to Brownian motion is negligible and P remains nearly constant against the fluid viscosity 1/. This
is attributed to the large diameter of the casein. The measurement, however, shows a noticeable
depolarization in the region approximately at 1/=50. Although not shown here, the particle diameter
was measured based on dynamic light scattering method (Otsuka electronics, ELSZ-2plus). The results
showed that the diameter largely decreases at approximately 1/=50. Further, the fluorescence intensity
increased markedly in the region of 1/>50. These results imply the possibility that the FITC (or a partof the casein cluster) has been detached from the casein at this condition. In this case, by having the
FITC, which has been concentrated on the casein, detached from the casein and apparently diluted in
the solution, the concentration quenching effect can be reduced and larger fluorescence intensity was
obtained. In addition to this, the separated molecule or cluster possess a smaller volume which decreases
the polarization degree.
Under the fluid viscosity condition of water, 1/1000~1200, P of C-FITC case shows a larger value
compared with those of the FITC case. This indicates that measurement of higher reliability andsensitivity can be performed by using the C-FITC as the fluorescence probe. The temperature
measurement discussed in the next section was conducted using the water solvent, and therefore, CFITC was used as the fluorescent molecule.
4.3. Fluid temperature effects
Results of temperature effects on fluorescence polarization are shown in Fig. 5. Measurement was
performed in the temperature range of 28 ~ 38°C, which is a reasonable range considering thetemperature applied to the biochemical assay. The symbols plotted in the figure are the average valuesof data measured for several times. The line drawn in the figure was derived on the basis of the leastsquare mean approximation using all measured data.
As shown in Fig. 5, the reciprocal value of fluorescence polarization 1/P increases linearly against
fluid temperature T. This agrees well the Perrin’s equation (Eq. (2)).
The empirical relationship between 1/P and T in the temperature range shown in Fig. 5 is presented
as Eq. (4).
1

−6.41

T= P0.139(4)

The sensitivity and accuracy of the present method was therefore 1.26%/°C and ±1.23°C,
respectively. These results confirms the reliability and feasibility of applying the fluorescencepolarization measurement to measurement of the fluid temperature.

7

7th European Thermal-Sciences Conference (Eurotherm2016)
Journal of Physics: Conference Series 745 (2016) 032097

IOP Publishingdoi:10.1088/1742-6596/745/3/032097

12.5
12.0

1/P

11.5
11.0
10.5
10.0
26

28

30

32

34

36

38

40

Temperature T [°C]

Figure 5. Relation of fluid temperature T and reciprocal value of P in the cases of C-FITC
solutions. The solid line is based on the least mean square approximation.
5. Conclusion
Measurement was performed to study the feasibility and accuracy of the fluid temperature method using
the fluorescence polarization P measurement. pH quenching effects varied P by approximately 20%,
which was largely reduced compared with the measurement of the fluorescent intensities the variationof which was 300~1000%. Measurement of the fluid viscosity effects showed that the casein-FITCconjugated probe provides high performance in the polarization measurement. Using the casein-FITC
fluorescent molecules, the reciprocal value of P showed a good linear relationship with the fluid
temperature with the sensitivity and accuracy of 1.26%/°C and ±1.23°C. The results agreed well withthe theory and confirmed the reliability and feasibility of the present method.
Acknowledgments
This work was partially supported by Japan Society for the Promotion of Science KAKENHI Grant
Number 15H03931, SPIRITS (Supporting Program for Interaction-based Initiative Team Studies) andMicro/Nano Fabrication Hub in Kyoto Univ. of "Low-Carbon Research Network" funded by the
Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
References[1] Kim Y H, Yang I, Bae Y S and Park S R 2008 Biotechniques. 44 495.
[2] Debby D, Bluhm R, Habets N and Kurz H 1997 Sensor and Actuators. 58 179.
[3] Yoon S Y and Kim K C 206 Optics and Lasers in Engineering. 44 224.
[4] Guilbault G G 1990 Practical Fluorescence (Marcel Dekker Inc.)[5] Arik M, Celebi N and Onganer Y 2005 Journal of Photochemistry and Photobiology. 170 105.
[6] Song L Hennik E J, Young I T and Tanke H J 1995 Biophysical Journal. 68 2588.
[7] Perrin F 1929 Ann. de Physique. 12 169.
[8] Weber G 1971 J. Chem. Phys. 55 2399.
[9] Obare S O 2010 Sensors. 10 7018.
[10] Magde D 1999 Photochemistry and photobiology. 70 734.
[11] Fowlkes J D 2006 Measurement Science and Technology. 17 5659.
[12] Dalgleish D G 2012 Food Science. 3 449.

8