high-spatial-resolution surface-temperature mapping using fluorescent thermometry

communications

908

Fluorescence thermometry

DOI: 10.1002/smll.200700581

High-Spatial-Resolution Surface-TemperatureMapping Using Fluorescent Thermometry**

Peter Low,* Beomjoon Kim, Nobuyuki Takama, and

Christian Bergaud*

The characterization of temperature and thermal properties

is of particular importance in micro- and nanotechnology.

Considering the highly increased density of structures and the

increased power dissipation per unit area associated with

miniaturization, good thermal design is of great importance

for device reliability and performance. Locating hot spots, for

example, on a microelectronic circuit, can be of great value in

evaluating a design, optimizing the performance, and perfor-

ming failure analysis.[1,2] Apart from the industrial applica-

tions of micro- and nanoscale thermometry, fundamental

questions of the thermal behavior, for example, thermal

transfer at a scale comparable to the phonon wavelength,[3]

could be more effectively addressed with improved character-

ization tools. The common approach for mapping temperature

on the microscale is based on infrared microscopy, which relies

on the analysis of the thermal radiation that is emitted from

any material. IR microscopy is a well-established technique

and can be used with relative ease for temperature mapping on

large scales. However, the technique suffers from a diffraction-

limited resolution, giving it an optimal spatial resolution of

around 5mm.[2,4,5] Nanoscale scientists typically use scanning

thermal microscopy (SThM) for high-resolution measure-

ments. Since the invention of the scanning probe microscope

at the beginning of the 1980s,[6] several scanning probes for

thermal characterization have been developed. The thermal

probes used are generally based on either thermocouple or

thermistor elements.[7–11] Other approaches have proposed

bimaterial cantilevers or fluorescent particles as temperature-

sensing probes.[12–14] The highest spatial resolution obtained

[�] P. Low, Dr. C. Bergaud

LAAS-CNRS, Nanobiosystems Group

University of Toulouse

7 Av de Colonel Roche, 31077 Toulouse Cedex 4 (France)

Fax: (þ33) 561-336-208

E-mail: [email protected]; [email protected]

P. Low, Prof. B. J. Kim, N. Takama

LIMMS-CNRS, Institute of Industrial Science

The University of Tokyo

4-6-1 Komaba, Meguro-ku, Tokyo (Japan)

[��] The financial support of the National Agency for Research(program ANR/PNANO 2006, project NANOTHERMOFLUO ‘‘ANR-06-NANO-004’’) is gratefully acknowledged. We also acknowledgethe laboratories of Prof. H. Fujita, Prof. H. Toshiyoshi, and Prof. S.Takeuchi for their support regarding experimental equipment andcleanroom facilities. We also thank Corinne Vergnenegre atLAAS-CNRS for her support on optical power measurements.Finally, we acknowledge the VLSI Design and Education Center(VDEC) at the University of Tokyo for the use of their AdvantestF5112 E-beam lithography facilities.

� 2008 Wiley-VCH Verl

to this date using SThM lies below 50 nm.[7] The main

drawback with the scanning probes is the slow readout rate. In

addition, the probes have to make contact with the sample and

thereby provide a thermal bridge between the sample and the

SThM equipment. Therefore, it is not obvious that the true

sample temperature is measured. Other thermometry tech-

niques typically found in the literature rely on Raman

spectroscopy[15–17] and thermoreflectance microscopy.[18–21]

Both are noncontact and noninvasive techniques that are

capable of rendering submicrometer spatial resolution.

Raman spectroscopy is well suited for the characterization

of large temperature ranges from room temperature up to

above 1000 8C. The spatial resolution is determined by its laser

spot diameter, which can be as small as 500 nm using a

high-magnification objective.[16] Although it is capable of

point measurements at very small timescales, the rendering of

images is slow as it analyses only one point at a time at a slow

acquisition rate of 0.5 points s�1.[22] Due to the low-energy

phonon modes of metallic surfaces, Raman spectra are not

suitable for the measurements of temperature on metals.

Thermoreflectance microscopy relies on the fact that the

surface reflectivity of any material depends on its temperature.

By measuring how incoming light is reflected, temperature

information can be extracted. The method can be applied

either in the scanning mode or in the full-field mode. The

scanning mode provides a superior temperature resolution but

suffers from a slower readout rate. The main drawback with

thermoreflectance microscopy is its tedious calibration

process. The thermoreflectance coefficient, which has to be

known for each material, is not readily available in the

literature in most cases. Furthermore, the reflectance proper-

ties are sensitive to factors such as the illumination wavelength

and, in the case of microelectronic devices, the thickness of the

passivation layer.[19] Another important drawback of thermo-

reflectance is that it is not well suited for measurements in

liquid conditions. Measuring temperatures in liquid conditions

is of interest in lab-on-a-chip applications where the control of

fluid temperature during analysis is important, particularly

during the reaction and separation.[23] Potential future

developments in nanoscale thermometry in general may lie

in the use of carbon nanotubes filled with liquid gallium.[24,25]

However, due to the requirements for temperature readout,

the function of these unconventional temperature probes has

so far only been demonstrated inside the high-vacuum

chamber of a transmission electron microscope.

In this Communication, we present results of the use of

fluorescent thermometry for submicrometer temperature

mapping followed by comparisons of the experimental results

with a finite element (FE) model implemented in the software

COMSOL. The basic principle of fluorescent thermometry is

that by analyzing the temperature-dependent fluorescence

from a fluorophore, the temperature at the location of the

fluorophore can be determined. Fluorescent thermometry has

during the last few decades found numerous applications, for

example, in fiber-optic temperature measurements,[26] in the

heat-transfer analysis of turbulent flows,[29–31] for the mapping

of temperatures in microfluidic devices,[22,27,28] and for the

evaluation of the heating properties of micro electromecha-

nical system (MEMS) heaters.[32] Fluorescent thermometry

ag GmbH & Co. KGaA, Weinheim small 2008, 4, No. 7, 908–914

has the capability to work as a full-field technique and thus

map temperatures over large areas with high recording speeds.

As compared to earlier papers targeting, for example,

temperatures in microfluidic channels, this paper illustrates

a method to investigate specifically the temperature on the

surface of a sample. To realize this, fluorophores are confined

to the vicinity of a sample surface by depositing a layer of the

fluorophores directly on the surface without dissolution in a

liquid or a resin. Thus, the measured signal will originate

completely from the plane of interest. This is particularly

advantageous when investigating temperature gradients of

high spatial confinement. The alternative choice of using

fluorophores dissolved in a resin or a liquid for the

measurement of surface temperatures might introduce measure-

ment errors since the captured fluorescence would originate not

only from the surface of the sample and since the temperature

would be likely to show a gradient throughout the height of the

resin or the liquid. The main factor limiting the spatial resolution

of fluorescent thermometry is the diffraction limit. With the use

of visible light, this gives us a potential spatial resolution of less

than 500 nm. The experimental setup and the general principles

of the technique are shown in Figure 1.

When performing fluorescent thermometry, it is of great

importance to use a fluorophore that emits a stable fluores-

cence. It is essential that the fluorescence is not permanently

modified when exposed to changes in its environment. Earlier

Figure 1. a) Resistive heating in a metal micro- or nanowire is used in o

fluorescent thermometry using an EMCCD camera to capture fluorescent im

B molecules. Excitation of the molecules as well as observation of the e

objective. d) Local heating occurs when a current is applied to the samp

fluorescence intensity changes. In the case of Rhodamine B, the intensity

of the relation between intensity change and temperature change, a give

small 2008, 4, No. 7, 908–914 � 2008 Wiley-VCH Verlag

work by Mao et al. and Walker et al. has pointed out the

potential of semiconductor nanocrystals as stable probes for

fluorescent thermometry.[33,34] However, preliminary investiga-

tions in our group on the specific case of CdSe/ZnS nanocrystals

have suggested that these probes lack thermal stability and are

permanently damaged when exposed to thermal cycles.[35] In

this work, we have instead used the molecular dye Rhodamine

B, which possesses a strongly temperature-dependent fluores-

cence intensity.[22,28,29] The main drawback of this fluorophore is

its strong photobleaching, which implies that it quickly loses

fluorescence when exposed to an excitation light source. In order

to evaluate the usefulness of this fluorophore for temperature

measurements, its photostability as well as stability over thermal

cycles were investigated.

The fluorescence stability investigations were performed

on dried Rhodamine B layers deposited on top of our silicon

substrates, which contained passivated micro- and nanowires

for later use as local heating elements. In order to evaluate the

photostability of the dried Rhodamine B layer, the fluores-

cence was measured over time during continuous excitation at

different intensities. The excitation intensity was altered by

the application of neutral density (ND) filters in the light path.

Fluorescent images of the Rhodamine B covered surface were

captured and followed by analysis of the global intensity of

each image. The evolution of these global intensities as a

function of time is shown in Figure 2. Photobleaching was

ur case to achieve locally confined temperature changes. b) Setup for

ages. c) The sample is covered by a thin and dense layer of Rhodamine

mitted fluorescence is performed through a high-magnification dry

le. Depending on the local temperature change, the local emitted

decreases when the temperature increases. e) With precise knowledge

n change in intensity can be converted to temperature.

GmbH & Co. KGaA, Weinheim www.small-journal.com 909

communications

Figure 2. Depending on the excitation level, the Rhodamine B molecules

are bleached at different rates. The graph shows the photobleaching

rates at three different excitation powers. Each curve was

normalized with regard to its initial intensity value.

Figure 3. As the Rhodamine B was subjected to thermal cycles, the

fluorescence intensity decreased slightly. This indicates a certain level

of thermally induced damage, although some of the intensity decreases

may be caused by photobleaching. The effect is not large enough to

prevent the usage of Rhodamine B as a thermal probe, although it

decreases the achievable temperature resolution.

Figure 4. Calibration curve for the temperature dependence of

Rhodamine B in dry conditions.

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obviously quite elevated at strong excitation levels, while

being less pronounced for weaker excitation. It may thus seem

that the best approach is to use a very weak excitation

intensity. However, we should keep in mind that, since the

emitted fluorescence inevitably decreases with the excitation

intensity, low excitation levels result in poor signal-to-noise

levels of the measured fluorescence. Therefore, it is of interest

to use the highest excitation intensity where photobleaching

can be kept under control. For the following investigations,

ND filters with a net transmittance of 0.36% were applied,

equaling an optical power density of 0.24 W cm�2 at the

surface of the sample.

Thermal stability was investigated by cycling the tem-

perature of the entire sample between 25 and 70 8C using a

microscope heating stage and measuring the global response

of the Rhodamine B fluorescence. Due to the inability of our

heating stage to actively decrease the temperature, the cooling

phase required a considerably longer time than the heating

phase. Photobleaching was minimized by using a computer-

controlled shutter to excite the Rhodamine B only during

measurements. The results of these intensity dependencies

over thermal cycles are shown in Figure 3. The intensity

decreases slightly with the cycles. We believe that this is

mainly due to photobleaching occurring during the short

exposure times and while optimizing the focus. However, with

the current data, the possibility of a small degree of thermally

induced bleaching cannot be eliminated. For our further

studies, we decided to maintain the same excitation level in

order to maintain a satisfactory signal-to-noise level.

After investigating the photostability and thermal stability,

the calibration of the temperature dependence of the

Rhodamine B fluorescence was performed. The entire sample

was again heated by the microscope heating stage to impose

well-defined temperatures throughout the Rhodamine B

layer. The calibration curve shown in Figure 4 could be

obtained as global fluorescent intensities at several tempera-

tures between 30 and 70 8C were measured. It should be noted

www.small-journal.com � 2008 Wiley-VCH Verlag G

that this calibration curve, which was obtained in dry

conditions, is less pronounced than a similar calibration curve

obtained in liquid conditions.[22,28]

Having determined the optimal excitation intensity and

acquired a calibration curve in bulk measurements, we pro-

ceeded to investigate the measurement of temperature at a

local level. While the former temperature changes were

imposed at a global level, local temperature variations were

now induced by resistive heating in our heating elements.

The heating elements consisted of passivated metal wires of

widths ranging from less than 1mm up to 4mm. The length of

the wires ranged from 40mm to 80mm while the thickness

of the wires was kept at 40 nm in all cases. To minimize the

effects of photobleaching, the computer-controlled shutter

was again used to illuminate the sample only when performing

the fluorescent intensity measurements. Figure 5 shows the

experimental results obtained when resistively heating one of

our microwires. From the top to the bottom of the table, the

current applied through the wire is gradually increased.

Images representing the registered fluorescent intensities are

shown in the second column. The brightness of each pixel of

mbH & Co. KGaA, Weinheim small 2008, 4, No. 7, 908–914

Figure 5. Resistive heating in an 80-mm-long, 2-mm-wide, and 40-nm-thick wire. When a current is applied through the wire, local resistive heating

occurs due to the geometrical confinement at the wire. The top row shows the case where no current is applied. The next three rows show the cases

for gradually increasing currents of 10 mA, 15 mA, and 20 mA. The first column shows the applied current value; the second column shows

the Rhodamine B fluorescence images, the third column shows the temperature images after the conversion of the intensity changes, and

the fourth column shows the temperature along the middle of the respective wire.

the image represents the intensity of the Rhodamine B

fluorescence in that pixel. Due to a difference in reflectivity,

the observed intensity is somewhat higher on the passivated

metal surfaces than on the areas without metal. Since the

temperature measurement relies on the conversion of the

relative change in intensity, this effect has little influence on

the measurements except for the fact that the signal-to-noise

ratio becomes higher on top of the metal. We assume here that

the presence or non-presence of metal underneath the SiO2

passivation layer does not alter the temperature dependence

of the Rhodamine B intensity. As expected, due to the local

increase in temperature, the fluorescence intensity at the

vicinity of the wire is decreased when a current is applied, with

a stronger decrease for higher currents. Using the calibration

curve, the relative changes in fluorescence intensity were

converted to temperature values and are represented as maps

in the third column. The strong confinement of the

temperature increase is clearly visible. Finally, the fourth

column shows the temperature values along the middle of the

wire. It is observed that considerable noise is present in the

measurement. This is mainly caused by the relatively low

excitation intensity used, which forces the use of high gains in

the camera system in order to capture the fluorescence. In the


present state, we estimate the temperature resolution to range

from 5–10 8C for a submicrometer spatial resolution. On the

other hand, if we sacrifice spatial resolution, the temperature

resolution can evidently be increased. The resolution is subject

to optimization via several paths, as discussed below.

The measured profile of the temperature distribution along

the wire is remarkably flat, with a steep increase of the

temperature at the ends of the wire and a relatively constant

value between the two ends. The reason for this plateaulike

profile is likely due to the fact that the microwire was

positioned on top of bulk silicon, which has a relatively high

thermal conductivity. Consequently, significant thermal losses

to the underlying substrate occur. To gain further insight into

the thermal transfers along the microwire, we implemented an

FE model of our device using the software COMSOL. A nickel

microwire with geometries corresponding to the real sample

was created. The three-dimensional (3D) geometry was

simplified by cutting out and analyzing only the lower left

quarter of the volume. This was possible because of the sym-

metries along the two centerlines of our microwire. Further

simplifications compared to the real sample were performed at

the sample boundaries. The temperature at the bottom surface

of our sample was set to a constant temperature corresponding


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to the ambient temperature. This is analogous to placing the

real sample on a heat sink that continuously evacuates the heat

generated in the sample. On the top and the sides of the

modeled sample, we assumed loss of heat through the natural

convection of air. This convection was represented using a

heat transfer coefficient hair¼ 5 W m�2 8C�1. Resistive heating

was simulated for the same current values as those tested

experimentally. The results are shown in Figure 6. The profiles

of the simulated temperature distributions exhibit a strong

resemblance to the experimental results. The temperature

changes abruptly at the ends of the microwire, while it plateaus

along the middle of the wire. However, the simulated

temperature values are somewhat lower than the experimental

values. We believe that this discrepancy is partially attribu-

table to the boundary conditions set at the bottom surface. In

the real case, the heat dissipation through the bottom surface

was not sufficient to maintain it at ambient temperature.

Figure 6. Using the FE simulation software COMSOL, theoretical predicti

made. From the top row to the bottom, simulated temperature maps of

increased, the temperature increases while the profile of the temperatur


To gain further insight into the influence of the underlying

substrate, simulations regarding the thermal conductivity of

the substrate were performed. In Figure 7, the results of

resistive heating in microheaters on substrates with different

thermal conductivities are shown. In one case, the wire was

placed on a silicon substrate and it thus had a highly elevated

thermal conductivity (130 W mK�1). In another case, the wire

was placed on a hypothetical substrate with a thermal

conductivity that was 1000 times lower than that of silicon

(0.13 W mK�1). As a reference, the thermal conductivity of

glass is approximately 1.4 W mK�1and that of air is approxi-

mately 0.025 W mK�1. In both cases, the same boundary

conditions as those in the simulation above were used. The

temperature maps clearly show how the temperature change

remains more confined in the case with an elevated thermal

conductivity. Simultaneously, we also note that the current

required to attain a certain temperature (100 8C in this case) is

ons, particularly those regarding the temperature distribution, can be

the four cases presented in Figure 5 are shown. As the current is

e distribution remains virtually the same.

mbH & Co. KGaA, Weinheim small 2008, 4, No. 7, 908–914

Figure 7. a) In our current sample, the metal wire is positioned on top of a solid silicon substrate. The temperature distribution, as simulated

in COMSOL when a current of 30 mA is applied, is shown. b) If we place the metal wire on top of a substrate with lower conductivity such as glass

or plastic (instead of silicon), a temperature distribution such as that shown in this image is obtained according to the simulations in

COMSOL. In this case, a thermal conductivity value of 0.13 W mK�1 (as compared to 130 W mK�1 for silicon) and a current of 5 mA were used.

c) Temperature values along the middle of the two wires in (a) and (b). The solid line curve corresponds to (a) and the dashed line curve

corresponds to (b).

significantly lower in the case with low thermal conductivity.

This result is expected since the heat dissipation from the wire

is greater when the thermal conductivity of the underlying

substrate is higher.

In conclusion, we have specifically investigated the

potential of the Rhodamine B fluorophore and fluorescent

thermometry in general to investigate temperatures on

micrometer and submicrometer scales with a simple micro-

scope setup. The technique focused on the measurement of

temperature distributions on the surface of a sample. By

confining a high concentration of fluorophore at the vicinity of

the surface, only fluorescence from the plane of interest was

captured. An FE model was implemented to simulate the

resistive heating in a microwire. Simulated results agreed well

with experiments in terms of temperature distribution profiles,

while the absolute values presented some discrepancies.

For measurements on the submicrometer scale, it will be

necessary to further enhance the technique in order to

optimize the signal-to-noise ratios. Things that may be

improved include in particular the excitation lamp stability

and the temperature-control equipment for thermal cycling

and calibration. In addition, an automatic and fast focus

control would be advantageous because it would shorten the

exposure time required for each intensity measurement and

thus limit the photobleaching effect. Solutions for increasing

the signal strength may be to minimize the photobleaching rate

by protecting the fluorophores from oxygen, which generally

accelerates the bleaching effect. This can be achieved by

covering the fluorophore layer with a thin resin layer. With

regard to the fluorescent thermometry in general, it is of

interest to continue trials on alternative fluorophores possess-

ing strongly temperature-dependent fluorescence and higher

photostability than the Rhodamine B molecule, for example,

Fluorescein,[31] green fluorescent protein (GFP),[36] and rare-

earth nanocrystals.[37,38] Our future investigations on fluor-

escent thermometry will target the response time of nanowire

heating as well as the measurement of surface temperatures on

samples immersed in liquids.

Experimental Section

Microstructure fabrication: Micro- and nanostructures were

realized on top of a thermally oxidized silicon wafer. The thickness

of the oxide was 500 nm. The positive resist ZEP-520A7 (Zeon


corp.) was spincoated onto the wafer at 5000 rpm for 60 s and

patterned by electron-beam lithography. Nickel was subsequently

evaporated onto the resist to create a 40-nm-thick layer. Lift-off of

the nickel was achieved through immersion in the resist remover

solution ZDMAC (Zeon corp.) at 60 -C for 15 min. The nickel

structures were finally passivated through the sputter deposition

of a 70-nm-thick SiO2 layer.

Materials and solvents: Rhodamine B powder (dye content

�95%) was purchased from Sigma–Aldrich. The Rhodamine B was

dissolved in deionized water at a concentration of 100mM. In order

to deposit the Rhodamine B on the sample, a glass coverslip was

placed at a height of approximately 100mm above the sample

surface using doublesided tape as a spacer, thus creating a

narrow gap. The introduction of the Rhodamine B solution in the

gap was facilitated by the capillary forces. After introduction in the

gap, the liquid was allowed to evaporate overnight to leave a

dense layer of Rhodamine B molecules on the surface. The density

of the Rhodamine B molecules on the surface after drying and the

spatial uniformity of this density were not precisely investigated.

We have assumed here that variations in the Rhodamine B density

do not influence the temperature dependence of the fluorescence

intensity. Under this assumption, possible spatial variations in

the molecule density do not introduce measurement errors since

the density in each point of the sample stays constant over

consecutive measurements and since we measure the relative, not

the absolute, intensity changes. On the other hand, density

variations imply that the signal strength varies over the surface,

giving rise to variations in the signal-to-noise levels. In compar-

ison, the situation would be different if we used fluorophores

dissolved in a liquid. In that case, the molecule density at a

specific location might indeed be different at different points in

time due to the movement of the molecules throughout the liquid.

Fluorescence microscopy: An Olympus BX-51 fluorescence

microscope was used for all observations. Observations were

made through an objective with 60T magnification and a

numerical aperture of 0.90. For the excitation and observation of

the Rhodamine B fluorescence, a 100 W mercury lamp was used

as a light source and a U-MWIG3 filter set from Olympus was used

to transmit excitation wavelengths in the range of 530–550 nm

and to observe fluorescence wavelengths above 575 nm. The

excitation intensity was manipulated using different combinations

of the ND filters U-25ND25 (25% transmission) and U-25ND6 (6%

transmission) from Olympus. The total optical power transmitted


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to the sample surface was measured using a Newport 840 optical

power meter. Optical power densities were subsequently obtained

by dividing the total optical power with the illuminated surface

area. The inaccuracy of these values was about W10% originating

mainly in the difficulty to determine an exact value of the

illuminated surface area. Fluorescent images were captured on a

computer using a Cascade II:512 EMCCD camera from Photo-

metrics. Images were analyzed in MetaMorph software from

Molecular Devices Corp.

Thermal manipulation: Thermal cycles and temperature

calibration were performed using a microscope heating stage

from Tokai Hit Corp., allowing for a precision of 0.1 -C. Active

cooling was not possible with the heating stage and thus led to

slow cooling times. In order to induce local resistive heating in the

microstructures, a current was applied using a dc current source.

When measuring fluorescence during thermal manipulation, a

transmission of 0.36% of the light source, equaling a power of

0.24 W cmS2, was used to restrict the photobleaching to a low

rate. In order to further minimize photobleaching, a computer-

controlled shutter was used in order to excite the Rhodamine B

only during measurements. During the thermal cycling trials and

during the acquisition of a calibration curve, thermal stabilization

was allowed for 10 min after attaining each new temperature

before making a measurement. Finding a good focus required

some time since thermal expansion inevitably led to focus drift. At

each temperature, ten images were captured, each with an

exposure time of 100 ms.

Keywords:fluorescence . nanowires . resistive heating . thermometry

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mbH & Co. KGaA, Weinheim

Received: July 24, 2007Revised: December 30, 2007Published online: May 26, 2008

small 2008, 4, No. 7, 908–914

high-spatial-resolution surface-temperature mapping using fluorescent thermometry

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