Optical Measurement of Temperature in Tissue Culture Surfaces

under Infrared Laser Light Excitation at 800nm using a Fluorescent

Dye

Claire Lefort, David Moreau, Philippe Lévêque and Rodney O’Connor

XLIM Institute, CNRS, Limoges University UMR 7252, 123 avenue Albert Tomas, Limoges, France

Keywords: Optical Temperature Measurement, Infrared Laser Light Excitation, Evolution of Temperature, Fluorescence

of Rhodamine B.

Abstract: The use of infrared laser light (IRLL) for biomedical applications has gained momentum the potential

applications in humans. The use of IRLL presents some limitations due to the dangerousness of these

radiations when exceeding some safety thresholds depending on the target tissue. This position paper

describes step by step a well-known technical method usually applied for microfluidics but here applied for

the first time to measure the temperature and the heat evolution in a cell culture environment under IRLL

excitation at 800 nm. The measurement of temperature is based on the property of Rhodamine B (RhB), a

fluorescent dye whose fluorescence intensity decreases linearly with temperature increases, illustrated by

preliminary microscopic measurements of temperature in cell culture dishes containing RhB solution under

the IRLL excitation from 0 mW to 300 mW.

1 INTRODUCTION

Infrared laser light (IRLL) in the near infrared range

(NIR) has become an indispensable tool for biological

studies with multiple applications, including

multiphoton microscopy (Hoover and Squier, 2013),

photodynamic therapy (Li et al., 2009) and neural

stimulation (Richter and Tan, 2014). A recurring

question often appears in the context of IRLL

excitation of biological targets (Welch 1984): what is

the potential impact of an IRLL excitation on tissue

health with respect to hyperthermic stress, cell or

DNA damage? This issue is particularly important in

the case of a tool dedicated to a clinical use on

humans. The evaluation of the short term or long term

effects of IRLL excitation on tissues is a complex

problem, including a large number of parameters to

consider: excitation parameters (average power,

wavelength, repetition rate, duration of exposition

and power or energy density). The first consequence

of IRLL excitation to be considered is the

modification of local temperature in the exposed

tissue and the range of temperature increase obtained

(Liljemaln et al., 2013).

The effects of different exogenous heating

sources, such as the IRLL excitation, have been

investigated with wide variety of microscopy

techniques for mapping temperature at the single-cell

level (Baffou et al., 2014). Among them, several were

applied to cultured cell lines, as in the Fluorescence-

lifetime microscopy based cellular measurements of

temperature (Shang et al., 2013). Fluorescence

lifetime depends on other parameters like the pH, the

viscosity or the solvent of the target making this

method uncertain for temperature measurement

above a limit of 37°C (Okabe et al., 2012). The

anisotropy of the fluorescence from green fluorescent

protein (GFP) has also been used for intracellular

temperature mapping in cultured cells exposed to

focal and bulk liquid heating (Donner et al., 2012).

This technique is highly promising, but requires the

use of established cell lines stably expressing

fluorescent proteins, making it unsuitable for neurons

or primary tissue cultures that are difficult to

transfect. Fluorescent quantum dot sensors have also

been used for intracellular temperature measurements

induced by external heat sources in a single living cell

(Martinez Maestro et al., 2010) or the Er

-doped

nanoparticle spectrum (Vetrone, et al. 2010). Other

methods include use of the temperature sensitivity of

the fluorescence spectra of the rare earth EuTTA

fluorescence spectrum (Zohar et al. 1998) or the

Lefort C., Moreau D., Lévêque P. and O’Connor R..

Optical Measurement of Temperature in Tissue Culture Surfaces under Infrared Laser Light Excitation at 800nm using a Fluorescent Dye.

DOI: 10.5220/0005256300470052

In Proceedings of the 3rd International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS-2015), pages 47-52

ISBN: 978-989-758-092-5

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

doped silica nanoparticles containing these

substances (Yang et al. 2014). Despite the nanometric

size of these particles, their application are hindered

by their repartition in the sample and differential

uptake into organelles and membranes, perturbing

their biochemical properties. The pioneering studies

using NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) and

Laurdan showed the potential of small organic

fluorescent probes as optical thermometers in living

cells (Chapman et al., 1995). Indeed, it was shown

that NBD fluorescence lifetime recordings were

suitable for monitoring ultrafast phenomena as the

impact of short pulse microirradiation on single cells.

Unfortunately the temperature resolution provided by

this method was approximately 2°C.

Rhodamine B (RhB), is a fluorescent, water

soluble dye, with an absorption peak at 554 nm, an

emission peak at 576 nm, and a temperature

dependent fluorescence quantum yield (Ferguson et

al., 1973). This quantum yield decreases linearly with

an increase in temperature (Kubin et al., 1982). When

the temperature is reduced, the value of the quantum

yield of spontaneous luminescence sharply increases

and makes certain that any possible negative effects

due to intermolecular relaxation processes are

overridden (Ali et al., 1990). RhB is highly

temperature sensitive, negligibly pressure sensitive,

and nominally pH independent above a pH of 6 (Shah

et al., 2009), and has been used to measure the

temperature in a wide variety of fluidic applications

(Shah et al., 2007; Gui et al., 2008; Ross et al., 2002;

Low et al., 2008; Sakakibura et al., 1999).

Interestingly, RhB has been also used as a

mitochondrial probe for measuring mitochondrial

membrane potential in cells (Reungpatthanaphong et

al., 2003). This work suggested that RhB can

distribute across biological membranes in response to

the transmembrane potential. This property lead us to

think that RhB dye could be useful in making

temperature measurements on small biological

compartments at the subcellular level. The sensitivity

of this technique was shown in the measurement of

temperature in rat tail tendon samples exposed to

radiofrequency electromagnetic fields (Chen et al.,

2009), and used to rule out any mean temperature

increases in cells exposed to nanosecond pulsed

electromagnetic fields (Kohler et al., 2013).

In this position paper, we present preliminary

work describing the method for the temperature

measurement in microscopic environment relevant to

the culture of biological cells under IRLL excitation

at 800 nm using the temperature-sensitive fluorescent

dye RhB. The calibration procedure is detailed and an

application for the measurement of temperature of a

cell culture surface containing RhB under an IRLL

excitation at 800 nm from a Titanium-Sapphire laser

delivered through an optical fibre is shown.

2 EXPERIMENTAL SETUP

The experimental setup is composed of two distinct

parts located in a temperature experimental room at

23.0°C. The global setup is presented in Figure 1.

Figure 1: Setup schematic for temperature measurements of

a RhB solution after heating with IRLL excitation from a

commercial Titan Sapphire laser system.

2.1 Setup for RhB Level of

Fluorescence Measurement

The local heating of the solution of RhB was tested

with a concentration of 50 µmol/L. The whole

experimental setup was placed inside a wide field

fluorescent microscope coupled with a light-emitting

diode (LED)-based light engine (Spectra7,

Lumencorp) and a combination of dichroics and

emission filters (Chroma or Semrock). The light from

the LED at 549 nm with 50 mW of average power

was focused at the surface of a petrie dish containing

RhB solution by a 20x microscope objective. The

backward emitted fluorescence from RhB was

collected in epifluorescent detection, with an electron

multiplied CCD with 512x512 pixels registered at 16

bits precision (Evolve 512, Photometrics). Both the

LED and CCD were controlled and synchronized by

a computer using freely available software (Winfluor,

Strathclyde University). The LED excitation and the

resulting RhB fluorescence emission were thus

measured at a frame rate of 0.198 s.

PHOTOPTICS2015-InternationalConferenceonPhotonics,OpticsandLaserTechnology

2.2 Conversion of the RhB

Fluorescence Intensity into

Temperature

The sensitivity of RhB fluorescence in measuring

temperature was previously shown to be 0.2°C

(Kohler et al., 2013) using two calibration steps. The

first calibration was made using a fibre optic

thermometer (Luxtron) with analogue voltage output

that was sampled with an analogue to digital board

(USB-6229-BNC, National Instruments) interfaced

with Winfluor, measuring the temperature of a dish of

water progressively heated with a metallic ring

containing heating resistance (Warner Instruments).

This step evaluated the linear relation between the

temperature of the target, measured with the

thermometer, and the voltage output of the fibre

thermometer, as read by Winfluor. Figure 2a presents

the evolution of the water temperature in the dish and

the voltage recorded by software. This defined the

calibration curve of the thermometer. Next, the petri

dish was filled with 2.5 ml of RhB solution and its

temperature was manipulated by the heating

resistance chamber. For each steps of 0.198 s when

the RhB solution temperature increased, the

fluorescence level was recorded. Figure 2b shows the

evolution of the temperature as measured with the

level of fluorescence detected by the CCD pixels with

a grayscale encoded on 16 bits.

Figure 2: a. Calibration of temperature measured by the

fibered thermometer as a function of its voltage measured

by Winfluor. b. Calibration of the temperature as a function

of the level of RhB fluorescence encoded in a grayscale on

16 bits measured by the CCD interfaced with Winfluor.

As expected, the level of RhB fluorescence decreased

linearly with the increase of the temperature of the

RhB solution. All the further results of measurements

will be expressed using this conversion of

fluorescence to temperature.

2.3 Infrared Laser Light Excitation

The focal excitation of temperature in the RhB

solution was realized by a femtosecond infrared

Titan-Sapphire (Ti: Sapph) laser. As this system is

very bulky, the IRLL was injected into a 10-m-long

fibre deporting the laser source wherever required in

the experimental room. The single mode fibre (SMF)

from Fibercore (reference SM750) was chosen for

delivering the IRLL with a mode field diameter of 5

µm and a numerical aperture (NA) equal to 0.12.

Figure 1 shows the whole experimental setup.

The IRLL came from a Ti: Sapp laser

(Chameleon, Coherent, INC.) delivering 140 fs pulses

at 800 nm, 10 nm of spectral bandwidth at the full

width half maximum (FWHM) at the repetition rate

of 80 MHz. Before the SMF input, an acousto-optical

modulator was inserted (Figure 1), allowing the rapid

modulation of average power before the IRLL

injection into the SMF. At the SMF output, the

maximum of average power stabilized at around 300

mW. After the propagation through a 10-m-long

fibre, the spectrum was broadened from 10 nm

FWHM to 100 nm FWHM resulting from nonlinear

effects and the pulse duration was increased from 140

fs to 10 ps due to dispersion effects. In the first

instance, in the context of the measurement of

temperature evolution of a solution of RhB, the

femtosecond properties of the pulses were not a

critical point in this experiment, although it might be

relevant and pursued in future experiments on

biological sample in the condition of imaging with a

two photon microscope.

3 RESULTS AND DISCUSSION

The optical fibre was placed 1 mm above the bottom

of the petri dish containing the RhB solution. Three

series of experiments were performed to illustrate the

use of RhB as a fluorescent dye allowing the

temperature measurement of a solution in response to

IRLL heating. The first experiment was carried out

with a continuous IRLL excitation. Next, the effect of

a periodic, intermittent IRLL excitation on RhB

fluorescence was investigated. Finally, the evaluation

of the temperature distribution of the RhB solution in

2D was studied in the cone of IRLL from the optical

fibre.

3.1 First Experimental Determination

of the Temperature Increase of a

Solution of RhB under a

Permanent IRLL Excitation

For the first set of experiments, the RhB fluorescence

was recorded on an area containing the entire region

illuminated by the IRLL. Figure 3a represents the

position of the optical fibre realized when delivering

few microwatts of IRLL at 800 nm, insufficient to

changing the RhB solution fluorescence. The

OpticalMeasurementofTemperatureinTissueCultureSurfacesunderInfraredLaserLightExcitationat800nmusinga

FluorescentDye

recorded image is shown in pseudo-colour without

the IR filter (Figure 1) in front of the CCD. This step

was dedicated to the fibre positioning in the field of

view of the CCD camera and the physical

delimitation of the ROI and selection of fluorescence

measurements by Winfluor. No LED excitation was

used to achieve this picture.

The objective of this part was to determine the

averaging of temperature in this ROI, resulting from

the continuous IRLL excitation at 800 nm over a total

duration of one min. This first experiment was done

with an average power of IRLL excitation of 200 mW

at the fibre output. Figure 3b shows the resulting

temperature measurement, determined after the

conversion of fluorescence to temperature, as

previously described.

Figure 3: a. Localisation of the optical fibre and ROI

delimitation of the measured level of fluorescence under

200 mW of IRLL excitation. b. Resulting temperature

measured in the ROI, blue: temperature measured without

excitation, red: temperature measured under the IRLL

excitation. c. Temperature measured in the ROI with 100

mW of IRLL excitation using exposure time of 10 min.

First, to compare the local heating resulting from the

IRLL excitation with a reference, we have measured

the temperature in the ROI without IRLL (Figure 3b,

3c blue curves). Then, the temperature of the ROI

under IRLL excitation was measured and is shown on

the same graph representing the temperature

measurement without IRLL. The fluorescence

measurements, switching on the LED at 549 nm, were

started after 2 s of recording, so as not to miss a

starting event. Then, the IRLL excitation thought the

optical fibre was started 3 s after the start of

fluorescence acquisition.

When the IRLL was started, the modification of

temperature was immediately visible and

characterized by the strong increase in the red curve

compared to the blue one (Figure 3b, 3c). In Figure

3b, the rapid deviation in the curve represents a

temperature increase of 1.7°C with a rise time less

than the 0.192 s between each point of the measure.

In the next seconds and until the half of the measure,

the local temperature increases until asymptoting to

26.3°C. The temperature was then stabilized at

26.5°C until the end of the recording. The decline of

the increase in temperature reveals the presence of a

steady state reached, illustrated also with the Figure

3c with an illumination duration of excitation of 10

and 20 min, respectively. The limitation in

temperature increase presumably results from the

large volume of RhB solution of 2.5 mL in the petri

dish, compared to the small volume under IRLL

excitation. By a simple calculation considering the

optical fibre NA, the index of the medium equal to

1.33 and the distance of 1 mm between the fibre tip

and the bottom of the dish, only 8.6.10

-6

mL of the

RhB solution was heated by IRLL excitation which

represents less than 0.01% of the total volume.

Therefore, an averaging of the temperature of the

ROI, by temperature exchanges with the whole

solution of RhB, are limiting focal elevation in

temperature. Further, the constancy in the

temperature especially in the case of the 10 min of

IRLL excitation, suggests that there is not a

significant photobleaching effect in the limit of the

duration of the measurement.

3.2 RhB Fluorescence Characterization

under an Alternative IRLL

Excitation

The objective was to investigate the possible effects

of an intermittent IRLL excitation compared to a

permanent illumination. The experimental protocol

implements three phases of 1 min of fluorescence

recording in the same ROI as the one defined in

Figure 3a. During the first phase of 1 min, the RhB

fluorescence without IRLL was recorded in order to

have a reference value. Next, the RhB fluorescence

was measured under IRLL excitation with an average

power of 150 mW. Finally, the influence of

alternating between IRLL excitation at 150 mW and

switching off was studied.

Figure 4: Intermittent IRLL excitation in the RhB

fluorescence. Blue line: without IRLL. Red line: constant

IRLL excitation. Green line: intermittent IRLL excitation.

The points of start and stop of the IRLL excitation are

explicitly identified. Globally, during the intermittent

PHOTOPTICS2015-InternationalConferenceonPhotonics,OpticsandLaserTechnology

IRLL excitation on and off (Figure 4 green curve), the

RhB solution was excited with IRLL for a total of 38

s. Different durations of IRLL excitation were applied

from 5 s until 18 s. The delay between two IRLL

excitations was constant and set at 6 s. The green

curve is alternatively very well merged with the red

line and with the blue line, respectively,

corresponding to the IRLL excitation or the absence

of IRLL (Figure 4). When the IRLL starts, in the

second and third start of excitation, the time necessary

for the green curve to be superimposed with the red

one was 1.5 s. In the case where the IRLL stopped,

the time delay for the RhB fluorescence was higher

than for the start. For the first stop, it was 3 s; for the

second, it was 3.6 s, as it was for the third one. These

long times of stabilization of more than 1 s, regardless

of high or low state status, may result from the

relatively small volume of the IRLL excitation highly

sensitive to temperature exchanges between the

excited volume and the whole volume of RhB

solution with a constant temperature equal to the

room temperature at 23°C.

3.3 Temperature Mapping

The optical fibre delivers the IRLL vertically as a

cone of light, without focusing or collimation of the

beam. The apex angle of the cone depends

exclusively on the numerical aperture of the fibre core

and the index of the medium approximated at water

at 1.33. The excitation is therefore spatially non

uniform leading to a not homogenous temperature of

the RhB solution in the whole petri dish. Thus, as the

level of RhB fluorescence is measured on a 2D plane,

a decrease in temperature distribution was observed

when moving away from the centre of the IRLL

illumination cone. Figure 5a details the position of the

6 ROIs. All of the 6 ROIs was touched together in a

line. Figure 5b reveals the resulting measurement of

temperature mapping in the 6 ROIs with and without

excitation.

Figure 5: a. Positions of the 6 ROIs in the cone of light from

the optical fibre; 6 areas in a line with a common limit. b.

Temperature mapping inside the cone of IRLL with and

without excitation. Inset: temperature evolution in the 6

ROIs between 0.90 and 0.95 min.

For all the measurement with or without IRLL,

each of the 6 ROIs fluorescence levels were recorded

in the same time. The first record without IRLL has

defined the reference temperature at 23°C. Then, the

IRLL was used to heat the target cone of RhB solution

with an average power of 70 mW. The temperature of

the 6 ROIs followed the same evolution law during

the rise time. During the following time, and until the

end, the temperature was stabilized, similar to that of

the first experiment (Figure 3c and 3d).

4 CONCLUSIONS

We have presented a method for measuring the local

temperature of a biological sample thanks to an

optical method using the temperature sensitive dye

Rhodamine B. The experimental setup was built

around a fluorescence microscope, with a light-

emitting diode illumination system for measuring the

level of fluorescence of RhB. We have first shown the

immediate influence of the infrared laser light

excitation at 800 nm at 200 mW caused a focal

increase of temperature of 1.7°C and the process of

temperature stabilisation for a recording duration

between 1 min and 10 min. Then, we have shown that

the fluorescence of the RhB can be also used in the

case of an intermittent IRLL excitation, with a good

reproducibility. Finally, the distribution of

temperature inside the cone of infrared laser light was

measured.

The important interest of this setup lies in its

simplicity of implementation, first requiring RhB, a

very standard and sensitive fluorescent dye with a

high sensitivity to temperature changes and resolution

of 0.2°C. Then, the insertion of the global setup for

temperature measurement around a fluorescent

microscope requires few simple modifications of the

microscope itself. Finally, as biological observations

are often made using fluorescent microscopy, the

additional tool of temperature analysis allows the

correlation of biological processes with the

modification of temperature.

For our future experiments, we will test this

method on the temperature modification of cells

under the effect of IRLL. The influence of

illumination and temperature on cell properties like

plasma membrane permeability under IRLL

illumination will be used to define a threshold of

optical parameters of the excitation not to be

exceeded to maintain cell viability in experiments.

OpticalMeasurementofTemperatureinTissueCultureSurfacesunderInfraredLaserLightExcitationat800nmusinga

FluorescentDye

ACKNOWLEDGEMENTS

This work has been highly supported by the LABEX

program of XLIM Institute in the axis “Biophysics

and health”, the Limousin Region and Limoges

University for the transversal thematic “Bio-Electro-

Photonic”. We also thank Fibrecore Inc. for providing

30 meters of the single mode fibre for free.

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PHOTOPTICS2015-InternationalConferenceonPhotonics,OpticsandLaserTechnology