Proceedings of the 17th International Heat Transfer Conference, IHTC-17
14 – 18 August 2023, Cape Town, South Africa
IHTC-17 | ID: 158

MEASUREMENT OF NEAR-WALL LIQUID TEMPERATURE
BASED ON TOTAL INTERNAL REFLECTION
FLUORESCENCE MICROSCOPY
R. Kuriyama1*, K. Ueda1, K. Tatsumi1 and K. Nakabe1
1

Kyoto University, Kyotodaiagku-katsura, Nishikyo-ku, Kyoto 615-8540, Japan

ABSTRACT
For the development of a measurement technique for near-wall liquid temperature, the present study
proposes two methods based on total internal reflection fluorescence microscopy (TIRFM). One is basedon two-color laser induced fluorescence (LIF) which exploits the temperature dependence of the intensity
ratio of two fluorescence bands from a single fluorescent dye. The other is based on fluorescencepolarization (FP) which exploits the depolarization phenomenon due to the rotational Brownian motion offluorescent molecules. In these methods, evanescent wave was generated at the interface between glass walland solution in a microchannel, so that only the fluorescent molecules near the wall within ~100 nm were
excited. In two-color LIF method using sulforhodamine B (SrB), the calibration result showed that thefluorescence intensity ratio of 580590 nm to 612644 nm decreased with temperature in the range of2540 C. However, the intensity ratio also decreased with time, which is presumably caused by theadsorption of SrB to the glass wall. In FP method using uranine, polarization degree decreased withtemperature in the range of 2540 C with the sensitivity of 0.7%/C, which agrees with the theoreticalvalue qualitatively. Temperature distribution measurement was also conducted using FP method in thevicinity of the channel bottom wall on which an Au line heater of 200 nm thick and 20 m wide waspatterned. The measured and calculated temperature distributions of the near-wall liquid agreed well within
1C in the area more than 50 m away from the heater in the spanwise direction.

KEY WORDS: Liquid temperature measurement, Near-wall region, Evanescent field, Two-color LIF,
Fluorescence polarization

1. INTRODUCTION
Thin liquid layer at the solid-liquid interface plays a crucial role in heat transfer phenomena both insingle-phase and two-phase flows. In single-phase liquid flow in micro- and nanochannels, for example,
the no-slip thermal boundary condition may fail due to the scale effect. This results in the temperature
jump at the boundary of liquid and the heating surface, which has significant effect on the heat transfer
characteristics as the channel scale decreases [1,2]. In gas-liquid two-phase flow, the dynamics of a thinannular liquid film which is driven by forced gas or vapor flow attract interest for efficient thermal
management with high heat flux [3,4]. The temperature gradient in liquid-gas interface generates
thermocapillary force which induces convection and rupture of the film. Also in nuclear boiling,
evaporation of a thin liquid film called microlayer, which is formed under a bubble growing on a heating
surface, is directly related to the bubble growth or shrinkage [5,6]. For understanding and controllingthese complicated heat transfer phenomena, advanced experimental techniques have been applied to thesolid-liquid interface. For investigating thermal slip length at the interface, time-domain
thermoreflectance [1,7] has been used to obtain thermal conductance of the interface. Surface-mounted
MEMS sensors [8,9] and IR thermography [10,11] have been employed as powerful tools for heattransfer measurements in boiling phenomena. However, it is still difficult to directly visualize twodimensional temperature distribution in liquid layer with submicron spatial resolution.
*Corresponding Author: kuriyama.reiko.2m@kyoto-u.ac.jp

For an alternative approach, total internal reflection fluorescence microscopy (TIRFM) which utilizes
evanescent field can be a candidate for a surface-specific and non-contact observation with high spatial
resolution. Although the most common applications of TIRFM are in cell biology and biochemistry,
TIRFM can also be applied to the diagnostics of thermal flow fields [12-14]. Guasto et al. [13] measured
liquid temperature in the region within 200 nm from a microchannel wall by using temperature sensitive
CdSe/ZnS quantum dots excited by evanescent field. They employed a ratiometric thermometry using
a temperature-insensitive rhodamine 110 dye to compensate the spatial distribution of the illumination
and realized an accuracy of 4.9 ºC. Kim and Yoda [14] performed temperature measurements within
300 nm from the channel wall using evanescent field and a temperature-sensitive fluorophore(fluorescein). They used an indium tin oxide film heater deposited on the bottom wall of the minichannel
to generate temperature gradients and the measurement results were in good agreement with thenumerical simulation within 0.65 ºC. Although the influence of using dye/quantum dots on the surface
properties (such as interfacial tension) requires careful consideration, these TIRFM-based techniques
are expected to have high potential to analyse various near-wall heat transfer phenomena.
In the present study, we propose two imaging techniques for near-wall liquid temperature based on
TIRFM. One is based on two-color laser induced fluorescence (LIF) which exploits the temperaturedependence of the intensity ratio of two fluorescence bands from a single fluorescent dye. The other isbased on fluorescence polarization (FP) which exploits the depolarization phenomenon due to therotational Brownian motion of fluorescent molecules. These methods determine the temperature by
ratiometric procedure based on intensity ratio or polarization degree which is normalized by the total
fluorescence intensity. Therefore, it is expected that the methods are robust to spatio-temporal variationin fluorescence intensity caused by various factors such as variations of excitation intensity andfluorophore concentration, and fluorescence degradation. This paper experimentally investigates thetemperature dependences of the intensity ratio and polarization degree of the fluorescence induced byevanescent field to examine the feasibility of the proposed methods. We also tried to visualize liquid
temperature distribution in the vicinity of the channel wall on which a line heater of 200 nm thick and20 m wide was patterned to generate temperature gradient.

2. MEASUREMENT PRINCIPLE
2.1 Total internal reflection fluorescence microscopy (TIRFM) TIRFM is an optical techniquewhich enables excitation of fluorescent molecules only in the vicinity of an interface by using evanescentfield. When light passes from one medium (with higher refractive index, n1) to another (with lower
refractive index, n2 (< n1)), some is reflected and the other is transmitted into the second medium. If theincident angle i is larger than the critical angle c (= sin-1(n2/n1)), all the light is reflected, i.e., it
undergoes total internal reflection. Even in this case, the electric field penetrates through the interface
and propagates parallel to the surface (in Fig. 1). This field is termed evanescent field. The intensity ofevanescent wave Ie (z) decays exponentially with the distance, z, from the interface as [15]:I e  z   I e  0  exp   z zp  ,(1)

zp  i 4 n12 sin 2 i  n2 2 .

(2)

where

z
Penetration depth

Evanescent fieldn2 ( n1)
n1
Incident lightzp
i

0 Ie(0)/e

Intensity

Reflected light

Fig. 1 Generation of evanescent field and its intensity distribution.

Ie(0) represents the intensity at the interface (z = 0). The characteristic length, zp, is called the penetration
depth and i is the wavelength of the incident light in vacuum. Since zp is generally on the order of i or
smaller, the evanescent field can selectively illuminate the near-wall area within ~100 nm from theinterface.

2.2 Two-color laser induced fluorescence (two-color LIF) LIF is often used to determine twodimensional distribution of scalar quantity (such as temperature, concentration, for example) based on
the dependence of fluorescence intensity on these quantities. The fluorescence intensity observed at awavelength  can be expressed as [16]:I f   ,T   A    I 0 Cf    ,T   L ,(3)

where A is the collection efficiency of the detection system, I0 is the intensity of the excitation light, Cf
is the concentration of the fluorescent molecule,  is the quantum yield, is the molar absorption
coefficient, and L is the length along the optical path of the excitation light. The quantum yield  istemperature dependent in most organic fluorescent molecules, whereas  does not have significant
temperature dependence. As a result, the fluorescence intensity changes with temperature, which has
the sensitivity typically on the order of 1%/ºC [17]. Based on this principle, the fluid temperature
measurement can be realized by measuring the fluorescence intensity. However, as seen from Eq. (3),the fluorescence intensity If is affected by various factors as well as temperature. Generally, excitation
intensity I0 and the concentration of fluorescent molecule Cf are not uniform over the measurement area
and can temporally fluctuate. To avoid the influence of these factors on the temperature measurement,
two-color LIF, which exploits the intensity ratio of two fluorescence bands from a single fluorescentdye, is promising. If the two fluorescence bands with different temperature dependences are spectrally
separated and simultaneously detected by two cameras, the ratio of the detected intensities, Rf, can be
defined as follows:Rf 

I f1 T 

I f2 T 

A11 T 

A2 2 T ,

(4)where If1 and If2 is the fluorescence intensities of the wavelength bands with high and low (or opposite)
temperature sensitivities, respectively. The intensity ratio Rf is independent of I0 and Cf, but it depends
on T through the ratio of the quantum efficiencies at two wavelength bands.

2.3 Fluorescence polarization (FP) When randomly oriented fluorescent molecules are irradiatedby a linearly-polarized excitation light, the probability of the absorption is proportional to the valuecos2, where  is the angle between the directions of excitation polarization and absorption moment of
the molecules. If the excited molecules are at stationary state, they emit the fluorescence polarized in
the same direction with the absorption moment. On the other hand, if the excited molecules are
suspended in fluid, they experience rotational Brownian motion during the fluorescence lifetime.
Consequently, the emission moments, which are parallel to the absorption moments, are randomized
according to the extent of their rotation. Depolarization of fluorescence is therefore observed with thedegree which is related to the fluid temperature, viscosity, and the molecular size.
Polarization degree, P, is defined as a function of I|| and I, which are the fluorescence intensities of thecomponents that are parallel and perpendicular to the excitation polarization, respectively:

P

I ||  I .
I ||  I (5)Since P is normalized by the total fluorescence intensity and follows the rotational motion of themolecules only, FP measurement is less influenced by the spatio-temporal fluctuation of I0, Cf, and 
compared to LIF measurement. Perrin [18] theoretically derived the equation which relates P to thefluorescence lifetime and the rotational diffusion of fluorescent molecules as:

(

1 1
1 1
kBT
 )  (  )(1 
 ),
P 3
P0 3
V

(6)

where P0 is the intrinsic polarization defined as the polarization degree of the molecule at stationary
state (in absent of rotation), kB is the Boltzmann constant, and V is the hydrodynamic volume of thefluorescent molecule. T and are the absolute temperature and viscosity of the fluid, respectively. From
Eq. (6), the reciprocal of P shows a linear relation with T/, if V and  remains constant during themeasurement. In the present study, FP measurement was conducted by using evanescent field
illumination generated with laser beam polarized perpendicular to the incident plane (i.e., s-polarized).

3. EXPERIMENTAL SECTION
3.1 Experimental setup Fig. 2 shows a schematic of the experimental setup for TIRFM-based
temperature measurements. A continuous wave laser (Coherent; Genesis MX488-1000SLM) operating
at 488 nm was used to generate evanescent field. The laser beam was introduced horizontally into a
trapezoidal prism (n = 1.522) mounted on a temperature control stage equipped with Peltier devices.
The beam travelled into a glass slide (silica, n = 1.463 or TEMPAX, n = 1.477) which serves as thebottom wall of a microchannel. An immersion oil (n = 1.518) was used between the prepared slide andthe prism to optically couple them. The beam underwent total internal reflection at the interface between
the glass and the solution in the microchannel and generated the evanescent field. The incident angle ofthe total internal reflection was 79 or 83º (depending on the glass material) and the penetration depth
was estimated to be ~70 nm from Eq. (2). The area where the evanescent field was generated had anellipsoidal shape with Gaussian intensity profile and the lengths of the major and minor axes of the area
were 5.3 mm and 1.2 mm, respectively.
The fluorescence was collected by an objective lens (Olympus; 10x, NA=0.25 in two-color LIF, 20x,NA=0.40 in FP), split into two components (If1 and If2 in two-color LIF, I|| and Iin FP), and measured
by two sCMOS cameras (Andor; Zyla 4.2 PLUS, 2048×2048 pixels, 16bit) simultaneously. The pixel
resolution of the images was 0.65 m/pixel in two-color LIF and 0.33 m/pixel in FP, respectively. In
two-color LIF measurement, a dichroic mirror (Semrock; FF506-Di03) and fluorescence filters(Semrock; FF01-628/32-25 and FF01-585/11-25) were used to spectrally separate the components. On
the other hand, in FP measurement, a bandpass filter (Chroma; AT535/40m) was used to extract thefluorescence and a polarizing beam splitter (Thorlabs; PBS251) was used to split the two polarizing
components.

3.2 Solution and microchannel In two-color LIF measurement, sulforhodamine B (Tokyo Chemical
Industry; A0600, SrB) was dissolved in a buffer solution of 7.010-3 mol/L sodium tetraborate (pH 9.2)
to suppress the adsorption to glass wall [19]. The concentration of SrB was set at 5.0  10-5 mol/L.
When excited by 488 nm, the fluorescence intensity of SrB decreases with temperature at 580590 nm,

MirrorMicrochannelzy45ºPolarizing
direction

PDMS
Glass slide

x

Prism

Dichroic mirror orpolarizing beam splitter

Objective lensCamera 1

Mirror

Bandpass filters
for two-color LIF

Bandpass filter
for FP

Camera 2

Laser

Intensity [a.u.]

Fig. 2 Schematic of the experimental setup for TIRFM-based temperature measurements.
580 590 612 644

60000
50000
40000
30000

25°C
30°C
35°C
40°C

20000
10000
0
500

550

600

650

700

Wavelength [nm]Fig. 3 Fluorescence spectra of SrB solution at different temperature conditions.

but it shows little change at 612644 nm as shown in Fig. 3. Thus, the fluorescence filters which transmit
these wavelength ranges were selected. The fluorescence measured through these filters were treated as
If1 and If2 in Eq. (4), respectively. In FP measurement, uranine (Nacalai Tesque, #3581692) was
dissolved in a buffer solution of 7.010-3 mol/L sodium tetraborate at the concentration of 1.010-4 mol/L
to obtain sufficient fluorescence intensity without suffering from concentration depolarization.
Fluorescent dye solution was supplied to a microchannel at a constant flow rate using a syringe pump
(Nihon Koden; CFV-3200). As shown in Fig. 2, the microchannel was comprised of polydimethyl
siloxane (PDMS) chip and a glass slide. The channel structure (width: 3 mm, height: 44 m) was moldedin a PDMS layer by soft lithography, and bonded to the glass slide by oxygen-plasma treatment. For FP
measurement, the glass surface was changed to be hydrophobic by silane coupling treatment using
commercially available glass coating agent (PPG; Aquapel) to minimize the adhesion of fluorescentmolecules. To monitor the channel bottom temperature, K-type thermocouples of 100 m in diameter
were inserted in 1-mm diameter holes of PDMS chip.

3.3 Experimental conditions The laser power at the measurement area (i.e., the glass-solution
interface in the microchannel) was estimated to be 160 mW based on the power measured just before
entering the prism and the reflection rate at the prism surface. The temperature of channel bottom wall
was controlled in the range 2540 C using the temperature control stage. The exposure time was set at
0.5 s in two-color LIF and 1.0 s in FP measurement, respectively. In both measurements, a pair of 100
successive images were obtained by two cameras. The averaged images of different color/polarization
components were registered to one another by using the affine transformation matrix which was obtainedby camera calibration. Then the distributions of fluorescence intensity ratio Rf or polarization degree P
were calculated using Eq. (4) or Eq. (6). It should be noted that the degradation (or fading) of thefluorescence on the fluorescence intensity measurement was negligible, since these measurements wereconducted in the presence of flow.

4. RESULTS AND DISCUSSION
4.1 Relationship between temperature and intensity ratio by two-color LIF measurement

Intensity ratio [-]Fig. 4 shows the result of three measurements. The temperature was increased from 25 C to 40 C with
5 C-increments and then decreased to 25 C with 5 C-decrements. The time and spatial averaged
values of the intensity ratio in the measurement area (1268 m585 m) are plotted against thetemperature with the neighboring number showing the measurement order. Although the intensity ratio
decreased with temperature as expected from the fluorescence spectra in Fig. 3, the temperaturesensitivity was higher when the temperature was increased (plot points #1#4) compared with thesensitivity when the temperature was decreased (plot points #4#7). When comparing the values at thesame temperature (plot points #1 and #7, for example), the intensity ratio measured later shows lower
value by approximately 5%. This trend can be observed in all three measurements in Fig. 4 and seems
related to the temporal change of fluorescence spectra. Fig. 5(a) shows the time variation of the intensity
ratio measured at constant temperature condition. The intensity ratio monotonically decreased with time
by 5% per hour and this is presumably caused by dye adsorption to channel wall. Fig. 5(b) shows thecomparison of the fluorescence spectrum of SrB solution flowing in microchannel (black line) and thatof SrB adsorped to the glass wall (red line). The peak wavelengths of these spectra are 588 nm and 581
nm, respectively, i.e., the spectrum of adsorbed SrB is slightly blue shifted compared that of solution.
This leads to the decrease in fluorescence intensity ratio with progression of dye adsorption over timeand complicates the temperature calibration. Also in the previous report by Kim and Yoda [14], where
they at first intended to measure near-wall liquid temperature by two-color approach using fluorescein
and SrB, the adsorptive characteristics of SrB prevented them from using it in the end. From these results,
it is suggested that some surface treatment should be employed to avoid adsorption when using SrB in
TIRFM-based measurement.
2.0
1.9

1
1
7

1.8
1.7

7
1
7

Ex. 1
Ex. 2
Ex. 3

2
3
6
2
6
2
6

4

5
3

4

5
3
5

4

35

40

1.6
25

30

Temperature [C]
Fig. 4 Relationship between temperature and the intensity ratio of fluorescence in 580590 nm to 612644 nm
nm from SrB solution obtained by two-color LIF measurement.
(b)
581 588

50000

1.95

Intensity [a.u.]

Intensity ratio [-]

(a)

1.90
1.85
1.80
1.75
0

15

30

45

Time [min]60

Adsorption
Solution

40000
30000
20000
10000
0
500

550

600

650

700

750

Wavelength [nm]Fig. 5 (a) Temporal change in the fluorescence intensity ratio and (b) comparison of fluorescence spectra from
SrB in solution and in absorbed state.

0.135
7

P [-]

0.130

1

0.125

Measured P
y=8.60×10-4x+0.153
6
2
3
5

0.120

4

0.115
25

30

35

40

Temperature [C]
Fig. 6 Relationship between temperature and the fluorescence polarization degree P from uranine solution
obtained by FP measurement.

4.2 Relationship between temperature and polarization degree by FP measurement Fig.
6 shows the measurement result obtained by FP method in the same manner as the previous section. The
plot point represents the time and spatial averaged polarization degree in the measurement area (520 m
 325 m) and the neighboring number shows the measurement order. The polarization degree P
decreased with temperature monotonically, which agrees with the theoretical prediction in Eq. (6). The
difference in the measured P value at the same temperature condition was within approximately 1%,
which was much smaller than the difference observed in two-color LIF measurement. The temperaturesensitivity of P was 0.7%/ºC, which was evaluated from the slope of the red straight line in Fig. 6 derived
by the least squares regression. The theoretical P values calculated for 25 and 40 C based on Eq. (6)are 0.017 and 0.012, respectively. There is large offset in the absolute values between theoretical andmeasured P values, which is mainly attributed to the transmission characteristics of the optical elements
such as dichroic mirror. The theoretical decrease of P between 2540 C is, however, on the same ordercompared with the measured value, which supports the validity of the measurement result.

4.3 Temperature distribution measurement by FP method For the validation of TIRFM-based
FP method, two-dimensional temperature distribution measurement was conducted in the near-wallregion of the straight microchannel where a temperature gradient was formed in the spanwise direction
by using an Au line heater. The width and height of the channel were 1 mm and 50 m, respectively,
and the uranine solution was supplied at the constant flow rate of 3 L/min. The heater was patternedon the surface of the glass slide along the longitudinal center line of the channel (i.e., patterned on thechannel bottom wall along the flow direction). The thickness, width and length of the heater were 200
nm, 20 m, and 15 mm, respectively, and the resistance was 137 at room temperature. The
microchannel was set on the prism mounted on the temperature control stage. The field of view (650 ×
650 m2) was set at the center of the channel in the spanwise and the streamwise directions. The
experiment was conducted in the following procedure. First, calibration experiment was conducted in
the microchannel in the same way as the previous section using the temperature control stage. Then thetemperature control stage was switched off and constant current of 40 mA was supplied to the heater.
The polarization measurement was conducted after the temperature reached steady state.
Fig. 7 shows the measurement result. The near-wall temperature distribution around the heater (shownin gray color) was clearly visualized. Although a fringe pattern aligned along the x-direction can beobserved, the fluid temperature is higher near the heater and gradually decreases with distance in the
spanwise direction due to the thermal diffusion. The measured temperature seems almost constant along
the x-direction. These trends qualitatively agrees with the temperature distribution obtained by thenumerical simulation (not shown here). In the calculated distribution, large temperature gradient wasobserved around the heater in the spanwise direction, whereas the temperature increase along the flow
direction was within 0.1 C in -300 m < x < 300 m. For quantitative comparison, Fig. 8 shows thespanwise distributions of the measured and calculated temperatures. The measured distribution in Fig.
7 was averaged over the x-direction and compared with the calculated distribution at x and z =0. These
two distributions agree within 1 C in the areas -50 m ≥ y and y ≥ 50m, but the measured temperature
is lower than the calculated one by 5 C in maximum in the areas |y| ≤ 50m. In addition, the temperature
profile is somewhat asymmetric about the y axis and shows lower value in the area y < 0 m, whichmight be caused by the misalignment of the Au line heater, i.e., the heater was not aligned perfectly
parallel to the flow direction. Although the exact causes of the fringe pattern and the large temperature
difference near the heater have not been understood yet, the above results indicate the potential of
TIRFM-based FP method for two-dimensional measurement of near-wall liquid temperature in channel
flow. We will further conduct experiments to examine the above issues and improve measurement
accuracy.

Temperature [C]

Fig. 7 Near-wall temperature distribution around the Au line heater (shown in gray color) measured by FP
measurement.
Electrode50
Experiment
Simulation

45

40

35
-150 -100 -50

0

50

100

150

y [m]
Fig. 8 Comparison of spanwise near-wall temperature distributions obtained by FP measurement and numericalcalculation.

6. CONCLUSIONSThe present study proposed TIRFM-based two-color LIF and FP for measurement of liquid temperaturein the near-wall region and investigated the feasibility of these methods by measuring the temperaturedependences of the intensity ratio and polarization degree of the fluorescence induced by evanescentfield. In two-color LIF measurement using SrB, the fluorescence intensity ratio of 580590 nm to612644 nm bands decreased with time by SrB adsorption to channel wall, as well as with temperature,
which made the calibration difficult. In FP measurement using uranine, the polarization degree
decreased with temperature in the range of 2540 C with the sensitivity of 0.7%/ºC. Using FP method,
non-uniform near-wall temperature field was visualized in the microchannel equipped with a line heater.
The measurement results agreed well with the numerical simulation within 1 ºC in the area more than
50 m away from the heater in the spanwise direction. We will further conduct temperature calibrationand distribution measurement to examine the measurement reliability and try to apply this method forthe measurement near three-phase contact line or in gas-liquid two phase flow in future work.

ACKNOWLEDGMENT
This work was partially supported by the Japan Society for the Promotion of Science KAKENHI, Grantin-Aid for Early-Career Scientists Grant Number 21K14095.

NOMENCLATURE
A
collection efficiency of system [-]T
Cf concentration of fluorescent molecule [mol/L] V
kB Boltzmann constant
[J/K]
x
L
Length along optical path[m]ynRefractive index[-]z
ILight intensity[W/m2]
zp
Ie
Intensity of evanescent wave
[W/m2]

2
If
Fluorescence intensity[W/m ]

I0
Intensity of excitation light[W/m2]
I||, I Fluorescence intensity of component parallel/ iperpendicular to excitation polarization [W/m2] PPolarization degree[-]
i
P0 Intrinsic polarization[-]

Rf Fluorescence intensity ratio[-]

Absolute temperature[K]
Hydrodynamic volume[m3]
Cartesian axis distance[m]
Cartesian axis distance[m]
Cartesian axis distance[m]
Penetration depth[m]
Molar absorption coefficient [L/(mol·m)]
Quantum yield[-]
Wavelength of light[m]
Wavelength of incident light in vacuum [m]Viscosoity[Pa·s]
Incident angle[rad]Fluorescence lifetime[s]

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