Proceedings
Proceedingsof
ofthe
theASME/JSME
ASME/JSME2011
20118th
8th Thermal Engineering Joint Conference
AJTEC2011
March 13-17, 2011, Honolulu, Hawaii, USA

AJTEC2011-44AJTEC2011-44461
MICROSCOPIC FLUID TEMPERATURE MEASUREMENTS
USING FLUORESCENCE POLARIZATION METHOD
Kazuya Tatsumi
Kyoto University
Kyoto, Kyoto, Japan

Akihisa Tozaki
Kyoto University
Kyoto, Kyoto, Japan

Kazuyoshi Nakabe
Kyoto University
Kyoto, Kyoto, Japan

ABSTRACT
A fluid temperature measurement method in microscopic
scale using fluorescence polarization is described in this study.
The present method has the advantages in not only noncontact
but also markedly reducing the influences of solution pH andquenching on the measured fluid temperature, compared toother methods including LIF (laser induced fluorescence)
method. In the case of a fluid at rest, the fluorescence intensity
varied with pH and linearly decreased with the elapsed time,
while the polarization degree remained nearly constant. The
polarization degree showed a good correlation with the fluid
viscosity and temperature that corresponded to the tendency of
the analytical results. The microchannel flows case also
showed a good correlation between the fluid temperature andthe fluorescence polarization degree was observed, which was
similar to the stationary fluid case. These results affirmed thefeasibility of our method using fluorescence polarization for
fluid temperature measurement.
NOMENCLATURE
C
concentration of fluorescent moleculesI
fluorescence intensity
flux of the excitation lightI0
I⊥
fluorescence intensity of the perpendicular component
fluorescence intensity of the parallel componentI//P
polarization degreettime [s]T
temperature [°C, K]
V
volume of the molecule [m3]Boltzmann constant [JK-1]
kB
εabsorption coefficient

FIGURE 1.

SCHEMATIC DIAGRAM OF THE MEASUREMENTPHYSICS USING POLARIZATION MEASUREMENT.

φ
λ
μ
τ
ξ

quantum efficiency
wavelength [m]fluid viscosity [Pa⋅s]
fluorescence life time [s]angle between the absorption moment and polarization
direction

INTRODUCTION
Fluid temperature control in microchannels is an important
issue to provide a suitable environment for cells and DNA, andto control the chemical reactions in the fields of medicine andchemistry. In order to control the fluid temperature in the1

Copyright © 2011 by ASME

microchannel, a method that can measure the fluid temperatureaccurately in microscopic scale is very important.
There are currently several methods that are typically used
to measure the fluid temperature in microchannels. Amongthese, thermocouples [1,2] and resistance thermometer [3] arerelatively simple and low-cost to carry out a point measurementof the fluid and wall temperatures. In these measurements,
however, a probe must be inserted in the channel that may
affect the fluid motion or the fluid temperature. Further, theinfluence of the heat capacity of the probes becomes more
apparent in micro-scale measurement that leads to a
deterioration of the measurement accuracy and resolution.
On the other hand, laser induced fluorescence (LIF)
method [4] derives the fluid temperature by measuring thefluorescence intensity of the fluorescent molecules mixed in thefluid. It does not require an insertion of mechanical probes in
the fluid, which means LIF can be applied as a noncontact
measurement. Another advantage compared with thethermocouples is its convenience in making a two-dimensional
measurement. The measurement principle of LIF method is thequenching effect of the temperature on the fluorescenceintensity, i.e. the fluorescence intensity decreases as the fluid
temperature increases. The fluorescence intensity, however, isalso influenced largely by the fluid pH, ionic concentration andmeasurement time. It is, therefore, difficult to subtract theseeffects from the temperature measurement during thecalibration experiment. Several methods have been suggested
to tackle these problems, such as using two types of fluorescentmolecules and exposing them under excitation lights withdifferent wavelength [5,6]. These methods however, requirecomplicate calibration measurements.
In this study, a fluid temperature measurement method
using fluorescence polarization is described. Figure 1 shows
the schematic diagram of the measurement method. The
fluorescent molecules are mixed in the working fluid, and are
exposed to a linearly polarized light. The emitted fluorescencefrom the molecules is also polarized in this case. However,having the molecule experiencing the Brownian rotation, apartial depolarization of the fluorescence will occur. In thiscase, the polarization degree of the fluorescence becomes afunction of the molecule volume, fluid viscosity, and the fluid
temperature. If a molecule with appropriate fluorescence
property and molecular size is chosen, the fluid temperature
can be obtained by measuring the polarization degree of thesuspended fluorescent molecules. The advantage of this method
is that the polarization degree will not be influenced by thefluorescence intensity, namely the fluid pH and otherquenching factors.
Measurements are carried out for the cases of stationary
fluid in a reservoir and flowing fluids in a microchannel. In thestationary fluid case, the effects of the fluid pH, elapsed time,and fluid temperature on the fluorescence polarization degree
are examined. Measurement is then carried out for
microchannel flows to evaluate the relationship between the

FIGURE 2. POLARIZATION DIRECTION OF THE EXCITATION
LIGHT AND FLUORESCENCE OF THE MOLECULES AT STATIC AND
BROWNIAN MOTIONS.

fluid temperature and polarization degree in order to verify thefeasibility and performance of the present method.
MEASUREMENT PHYSICS
As mentioned in the previous section, the present methodmeasures the polarization degree of the fluorescence emitted
from the molecules. The polarization direction of thefluorescence shows a strong relation with the polarization
direction of the excitation light and Brownian motion of themolecules. The fundamental principles of the measurement
method will be described in this section.
Figure 2 shows the relationship between the polarization
directions of the excitation light, fluorescence, and absorption
moment of the fluorescent molecule. As shown in Fig. 2(a), we
now consider a fluorescent molecule, of which the absorption
moment direction is parallel to the z axis and the position
located at the origin, exposed to a linearly polarized excitation
light. The absorption degree of the molecule to the excitation
light depends on the angle between the absorption moment andthe polarization direction of the excitation light, ξ. As ξ
increases, this absorption rate decreases in the rate of cos2ξ.
When the excitation light is polarized in the x direction, themolecule will not be excited. On the other hand, when the
excitation light is polarized in the z direction, the absorption
rate of the molecule becomes maximum (Fig. 2(a)).
The emitted fluorescence is also polarized in the same
direction with the absorption moment. In other words, if an
infinite number of fluorescent molecules randomly positioned
are exposed to a linearly polarized excitation light, themolecules with the absorption moment parallel to the excitation
light are stochastically excited. The fluorescence observed fromthese molecules is, therefore, mainly polarized in the same
direction with the excitation light.
When the molecules are suspended in the fluid, therotation of the molecule due to the Brownian rotation
significantly influences the polarization direction of thefluorescence in addition to the effects of angle ξ. That is, since
the molecule rotates during the period of the fluorescenceemission, the polarization direction of the fluorescence shows2

Copyright © 2011 by ASME

some variance with the direction of the excitation light (SeeFig. 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⊥ represent the fluorescence intensities of thecomponents that are parallel and perpendicular to thepolarization direction of the excitation light, respectively.
Perrin [7] and Weber [8] have theoretically derived Eqn. (2) toexpress the characteristic of P of a fluorescent molecule with
Brownian motion.
⎛ 1 1 ⎞ ⎛⎜ 1 1 ⎞⎟⎛ k BT ⎞τ⎟⎜ − ⎟ = ⎜ − ⎟⎜⎜1 +μV ⎟⎠⎝ P 3 ⎠ ⎝ P0 3 ⎠⎝FIGURE

3.

SCHEMATIC

VIEW

OF

THE

EXPERIMENTALAPPARATUS AND OPTICAL SYSTEM.

(2)

P0 is the polarization degree of the molecule in steady state(without rotation). kB is the Boltzmann constant. μ and T are theviscosity and temperature of the fluid, respectively. τ and V are
the fluorescence relaxation time and the volume of themolecule, respectively. As one can see in Eqn. (2), thereciprocal number of P shows a linear relation with the T/μ.
Therefore, if the molecular volume remains constant during themeasurement, the fluid temperature can be obtained bymeasuring the polarization degree of the fluorescence. The
present measurement is based on this physics.
The total fluorescence intensity I can be influenced byvarious factors. For example, I can be presented by Eqn. (2).
I = I0Cφε(2)

I0 is the intensity of the excitation light and C is theconcentration of the florescent molecules. φ is the quantum
efficiency, and ε is the absorption coefficient presenting the ratethat the excitation light is absorbed while it passes through aunit length of solution.
In addition to these effects, the decrease of fluorescenceintensity, so called a quenching effect, occurs attributed tovarious reasons [9]. For example, when the fluid temperature
increases, the molecular motion in the solution becomes active.
In this case, the fluorescent molecules collide with othermolecules that leads to a decay of the molecular energy, i.e. thedecrease of fluorescence intensity. The change of the fluid
temperature also produces a change in the vibration motion of
the molecule, hence, the fluorescence intensity. Fluid
temperature measurement using the laser induced fluorescence(LIF) method employs this relationship between the fluid
temperature and fluorescence intensity.
The fluorescence intensity is, however, also influenced by
the pH of the solution, particularly with molecules including

FIGURE

4.

SCHEMATIC

VIEW

OF

THE

RESERVOIRFORSTATIONARY FLUID MEASUREMENTS AND MICROCHANNEL.

acidic. Oxygen quenching that is attributed to the oxygen
molecules, which are solved in the fluid, colliding with thefluorescence molecules is also another typical quenching effect.
These other effects will inevitably influence the performance of
the LIF measurements, and are not preferable. Therefore,
several methods have been proposed in order to reduce theinfluence of these quenching effects, or to subtract such effects
from the original data.
On the other hand, the present method measures thepolarization degree, P. As shown in Eqn. (1), P is a value
normalized by the value I//+I⊥ that presents the total
fluorescence intensity. P is, therefore, not influenced by thevariation of the fluorescence intensity. This means that the3

Copyright © 2011 by ASME

2.5

previously mentioned quenching effects can be ignored in thepresent method.

I/I7.0o

I/I7.0
1.5

1.0
6.5

7.0

7.5

8.0

8.5

9.0

9.5

pH

10.0

FIGURE 5. pH EFFECTS ON FLUORESCENCE INTENSITY, I.
2.2

P/P7.0EXPERIMENTAL PROCEDURE
Figure 3(a) shows the schematic view of the experimental
apparatus used to measure the polarization degree P. Figure3(b) shows the schematic diagram of the optical system. The
apparatus is composed of a microscope (Olympus; BX-51),
photomultiplier (Hamamatsu; C7319) and data acquisition
equipment (National Instruments; USB-9219). A halogen lamp
is used as the light source. The light passes the band-pass filter(Semrock; FF01-482/35-25, λ=482±18nm) and a condenser, inwhich a polarizer is embedded, and is emitted to the test section
as the excitation light. The fluorescence is measured by thephotomultiplier through an objective lens (Olympus;
UMPlanFL, ×10, NA=0.30, working distance=10.1mm), a
band-pass filter (Semrock; FF01-536, λ=536±20nm) and arotatable analyzer (Olympus; U-AN360-3).
The polarization degree, P, was measured by the following
procedure. The angle of the polarizer installed in the excitation
pathway was fixed. The angle of the analyzer was calibrated forthe directions parallel and perpendicular to the polarization
direction of the excitation light without inserting the test
section in the optical pathway. For the fluorescencemeasurement, the components of the intensity that are paralleland perpendicular to the excitation polarization (I// and I⊥) aremeasured by rotating the analyzer during the measurement. P isthen obtained applying these values to Eqn. (1).
Measurements were made for two kinds of flow
conditions: one is a fluid at rest and the other is the fluid
flowing in a microchannel. Figure 4 shows the schematic
diagrams of the test section used in these measurements. In
Figs. 4(a) and (b) are shown the reservoirs prepared for thestationary fluid measurement. The reservoirs were 5mm
diameter circular cylinders made of PDMS and cupper,
respectively. For the cupper reservoir, water of constanttemperature was supplied from a thermostat to the cupper blockin order to maintain the wall and fluid temperature inside thereservoir constant. The fluorescence solution was sealed in thereservoir by attaching cover glasses to the top and bottom
walls. Black paint was sprayed on the surface of the cylinder
inner wall to reduce the reflections. K-type thermocouples wereembedded in the cupper block at the locations depicted in Fig.
4(b) to measure the wall temperature.
Measurement for the microchannel flow was carried out
using the test section shown in Fig. 4(c). The microchannel was
made of PDMS (poly-dimethylsiloxane, Shin-Etsu Chemical
Co. Ltd.; KE-106) and was fabricated using SU-8 (MicroChem
Co.) as a casting mold. The PDMS channel, which wasremoved from the SU-8, was attached to a cover glass. The
channel had a cross-section of 1mm width and 50μm height.
Fluid was supplied from a syringe pump (Nihon Kohden; CFV3200) to the channel with a flow rate of 0.5μl/min.
The microchannel was attached to the top wall of thecupper block with the channel center positioned at the center of

ΔT=5 C

2.0

2.0

P/P7.01.8

ΔT=5 C

o

1.6
1.4
1.2
1.0
6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

pH
FIGURE 6. pH EFFECTS ON POLARIZATION DEGREE, P.

the cylinder through which the excitation light was emitted. Ktype thermocouples were embedded aside the microchannel at
the locations illustrated in Fig. 4(c).
EXPERIMENTAL CONDITIONSAs mentioned in the above section, polarization
measurement was made for the stationary fluid and themicrochannel flow. The elapsed time during the measurement,
fluid pH, and fluid temperature were varied in order to evaluate
their influences on the polarization degree, P. For the stationary
fluid measurement, the influence of the pH was measured using
the PDMS reservoir shown in Fig. 4(a); a condition closer tothe one used in practical applications. For the measurement toevaluate the effects of the elapsed time and fluid temperature,
the cupper block type reservoir was used to control thetemperature. For the microchannel flow measurement, the fluid
temperature was varied using the microchannel attached to thecupper block as shown in Fig. 4(c).
Fluorescein-5-isothiocyanate (FITC) was used as the watersoluble fluorescence molecule. The molecular weight of the
FITC was MW=389. Considering the distribution of P against
the variable kBTτ/μV obtained from Eqn. (2), P reciprocallyincreases as the term kBTτ/μV decreases. This indicates that ahigher sensitivity is achieved in the region of larger P since thepresent method evaluates the temperature difference from thevariation of P. Considering then that P decreases as 1/V
decreases, a molecule with greater volume will enhance themeasurement sensitivity. For this reason, casein-FITCconjugated (ABD Bioquest; MW=13440, hereafter referred to4

Copyright © 2011 by ASME

1.05
1.00
0.95

I/It=0as C-FITC) is used in the measurement. Casein has a muchlarger molecular size than FITC, and is a protein that can bemarked easily by the FITC.
Phosphate buffer saline (Nakarai tesque) was used as thesolvent. C-FITC was mixed with the solvent at a concentrationof 0.01g/l (7.4×10-7mol/l). The pH of the solution was checked
before measurement using a pH sensor (Horiba; B-212). Except
the measurement evaluating the pH effect, the pH of thesolution was kept as pH=9.18.
The sampling rate of the data acquisition system was 2Hz.
5 samples were measured continuously and was averaged forthe I// and I⊥ components by rotating the analyzer. This process
was made alternatively 10 times, and the averaged value wastaken as the result. From the I// and I⊥ values, P was calculatedusing Eqn. (1).

0.90
0.85

I/It=00.80oΔT=1 C

0.75
0

20

40

60

80

100

t [min]FIGURE

7.

MEASUREMENT ELAPSEDTIME

EFFECTS ON

FLUORESCENCE INTENSITY, I.

P/Pt=0

1.00
0.95
0.90

P/Pt=00.85
0oΔT=1 C
20

40

60

80

100

t [min]FIGURE

8.

MEASUREMENT ELAPSEDTIME

EFFECTS ON

38

40

POLARIZATION DEGREE, P.
14.0
13.5
13.0

1/P

RESULTS AND DISCUSSION
Figures 5 and 6 show how the fluid pH influences thefluorescence intensity I and the polarization degree P in thestationary fluid case. The results are normalized by I7.0 and P7.0
that present the I and P values of pH=7.0, respectively. I/I7.0
shown in Fig. 5 takes a maximum value at pH≅9.0, anddecreases as pH increases or decreases. This result shows thequenching effect of pH on the fluorescence of C-FITC. As one
can see, the value at pH=9.0 is approximately 2.5 times larger
than the value of pH=7.0, i.e. I7.0.
On the other hand, as shown in Fig. 6, the value P/P7.0 is
nearly constant in the range of 7.0≤pH≤7.5, and then increases
in the area of pH=7.5~8.5. At pH greater than 8.5, P/P7.0
remains almost constant. The difference between the maximum
and minimum values is 5.5%: a much smaller value comparedto the I/I7.0 case in the corresponding pH region. The range ofthe vertical axes of the graphs shown in Figs. 5 and 6 are
adjusted so that the same variation in each graph will represent
the same amount of temperature variation (the ranges
corresponding to 5°C temperature difference in themeasurement are depicted in each figure). Therefore, thedifference in the variation in each graph will directly represent
the variation in the temperature measurement. This shows thatthe variation attributed to the fluid pH can be markedly
decreased in the polarization measurement that leads to higher
resolution and accuracy of the temperature measurement.
Figures 7 and 8 show the influence of the measurement
elapse time t on the I and P values. The results are normalized
by It=0 and Pt=0, that present the values at the starting time of themeasurement, respectively.
As shown in Fig. 7, I/It=0 gradually decreases as themeasurement starts. At time t =110min, I shows 20% reduction
compared with It=0. This is considered to be due to thequenching effects of various causes as described in theprevious section. P/Pt=0 in Fig. 8, however, remains nearly
constant during the measurement. Same as in Figs. 5 and 6, therange of the vertical axis in the two figures corresponds to thesame range of temperature variation. In this case, the variations

12.5
12.0
11.5

30

32

34

36 o

T[ C]

FIGURE 9. RELATION BETWEEN RECIPROCAL OF P AND FLUIDTEMPERATURE T IN RESERVOIR.

during the period of 0≤ t ≤110min for I/It=0 and P/Pt=0
correspond to the temperature differences of 10°C and 1.4°C,
respectively. The influence of time on the present method is,therefore, markedly small compared with the methods using thefluorescence intensity measurement.
Figure 9 shows the relation between the reciprocal of P
and the fluid temperature in the reservoir T. The line drawn in
the graph was derived on the basis of the least square mean
approximation using all plotted data. A good linear correlation
between 1/P and T is observed in the graph. The error bars
shown in the figure represents the results of uncertainty
analysis conducted on the basis of the ASME Performance Test

5

Copyright © 2011 by ASME

14.0

1/P

13.5
13.0
12.5

Microchannel
Reservoir

12.0
11.5

30

32

34

36

o

38

40

T [ C]FIGURE 10. RELATION BETWEEN RECIPROCAL OF P AND
FLUID TEMPERATURE T IN MICROCHANNEL.

Code [10]. As shown in the figure, the maximum error
corresponds to the temperature difference of approximately
0.49°C.
Figure 10 shows the relation between 1/P and thetemperature T of the fluid flowing in the microchannel. The
results of the stationary fluid case are also shown in the figure
for comparison. Same as the results shown in Fig. 9, 1/P
linearly increases against T, and a good correlation is observed.
This result affirms the feasibility of the present method fortemperature measurement in microchannel using fluorescencepolarization.
Comparing the results obtained from the stationary fluid in
the reservoir (Fig. 9) and the flowing fluid in microchannel(Fig. 10), the results of the latter case show larger 1/P value
and smaller slope in the least square mean line. This indicateslower measurement sensitivity. The reason for this isconsidered to be attributed to the optical quality of the PDMS
microchannel wall. Since the fluorescence was measured
through the PDMS, depolarization of the fluorescence might
have occurred for some degree as the fluorescence transmits.
This depolarization effect will be superimposed on that causedby the Brownian motion. Therefore, P is equally increased in
the overall range and the measurement sensitivity decreases.
In addition to this, an apparent increase in the error isobserved in the microchannel case. The reason for this isbelieved to be attributed to the decrease of the fluorescenceintensity. As shown in Fig. 4(c), the microchannel was attachedon the cupper block. The distance between the condenser andthe working fluid with fluorescent molecules, therefore,
increased that leads to a decrease of the intensity of theexcitation light. Further, the volume of the microchannel wasmuch smaller compared with the reservoir. Therefore, thenumber of the fluorescent molecules measured was muchsmaller the microchannel. For these reasons, the fluorescenceintensity decreased and the influence of the error in the opticalmeasurement increased in the microchannel case. These
problems can be solved by adjusting the focal position of theillumination and measurement parts, or increasing theconcentration of the fluorescence molecules.

CONCLUSIONS
A noncontact fluid temperature measurement inmicroscopic scale using fluorescence polarization was
described. The effects of pH, elapsed time and fluid
temperature on the measurement of fluid at rest andmicrochannel flow were discussed. A linear relation between
the polarization degree and fluid temperature was observed forboth cases. The pH and elapsed time had a considerable
influence on the fluorescence intensity whereas the polarization
degree was scarcely affected. These results showed thefeasibility and advantage in using fluorescence polarization for
fluid temperature measurement.
ACKNOWLEDGMENTS
This study was financially supported by The Japanese
Ministry of Education, Culture, Sports, and Science.
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