Fluorescence-based thermometry for precise estimation of nanoparticle laser-induced heating in cancerous cells at nanoscale

Abstract

Photothermal therapy (PTT) has attracted increasing interest as a complementary method to be used alongside conventional therapies. Despite a great number of studies in this field, only a few have explored how temperatures affect the outcome of the PTT at nanoscale. In this work, we study the necrosis/apoptosis process of cancerous cells that occurs during PTT, using a combination of local laser heating and nanoscale fluorescence thermometry techniques. The temperature distribution within a whole cell was evaluated using fluorescence lifetime imaging microscopy during laser-induced hyperthermia. For this, gold nanorods were utilized as nanoheaters. The local near-infrared laser illumination produces a temperature gradient across the cells, which is precisely measured by nanoscale thermometry. This allows one to optimize the PTT conditions by varying concentration of gold nanorods associated with cells and laser power density. During the PTT procedure, such an approach enables an accurate determination of the percentages of apoptotic and necrotic cells using 2D and 3D models. According to the performed cell experiments, the influence of temperature increase during the PTT on cell death mechanisms has been verified and determined. Our investigations can improve the understanding of the PTT mechanisms and increase its therapeutic efficiency while avoiding any side effects.

Full text

Nanophotonics 2022; 11(18): 4323– 4335
Research Article
Oleksii O. Peltek, Eduard I. Ageev, Pavel M. Talianov, Anna D. Mikushina, Olga S.
Epifanovskaya, Aliaksei Dubavik, Vadim P. Veiko, Kirill Lepik, Dmitry A. Zuev, Alexander S.
Timin* and Mikhail V. Zyuzin*
Fluorescence-based thermometry for precise
estimation of nanoparticle laser-induced heating
in cancerous cells at nanoscale
https://doi.org/10.1515/nanoph-2022-0314
Received May 31, 2022; accepted August 1, 2022;
published online August 15, 2022
Abstract:Photothermal therapy (PTT) has attracted
increasing interest as a complementary method to be used
alongside conventional therapies. Despite a great number
of studies in this eld, only a few have explored how
temperatures aect the outcome of the PTT at nanoscale.
In this work, we study the necrosis/apoptosis process of
cancerous cells that occurs during PTT, using a combina-
tion of local laser heating and nanoscale uorescence ther-
mometry techniques. The temperature distribution within
a whole cell was evaluated using uorescence lifetime
imaging microscopy during laser-induced hyperthermia.
For this, gold nanorods were utilized as nanoheaters. The
*Corresponding authors: Alexander S. Timin and Mikhail V. Zyuzin,
School of Physics and Engineering, ITMO University, Lomonosova 9,
191002, St. Petersburg, Russian Federation,
E-mail: alexander.timin@metalab.ifmo.ru (A.S.Timin),
mikhail.zyuzin@metalab.ifmo.ru (M.V.Zyuzin).
https://orcid.org/000-0002-5364-2635 (M.V.Zyuzin)
Oleksii O. Peltek,Eduard I. Ageev,Pavel M. Talianov,Vadim P.
Veiko and Dmitry A. Zuev, School of Physics and Engineering,
ITMO University, Lomonosova 9, 191002, St. Petersburg, Russian
Federation, E-mail: aleksey.peltek@metalab.ifmo.ru (O.O. Peltek),
eduard.ageev@metalab.ifmo.ru (E.I. Ageev),
pavel.talianov@metalab.ifmo.ru (P.M. Talianov),
vadim.veiko@mail.ru (V.P. Veiko), d.zuev@metalab.ifmo.ru
(D.A. Zuev). https://orcid.org/0000-0001- 5626-7677 (E.I. Ageev)
Anna D. Mikushina, Laboratory of Renewable Energy Sources, Alferov
University, Khlopina 8/3, 194021, St. Petersburg, Russian Federation,
E-mail: annamikushina303@gmail.com
Olga S. Epifanovskaya and Kirill Lepik, RM Gorbacheva Research
Institute of Pediatric Oncology, Hematology and Transplantation,
Pavlov University, Lva Tolstogo 6/8, 191144, St. Petersburg, Russian
Federation, E-mail: epif-olga@rambler.ru (O.S. Epifanovskaya),
lepikkv@gmail.com (K. Lepik)
Aliaksei Dubavik, Faculty of Photonics, Center of Optical Information
Technologies, ITMO University, Birzhevaya liniya 4, 199034, St.
Petersburg, Russian Federation, E-mail: adubavik@yandex.ru
local near-infrared laser illumination produces a tempera-
ture gradient across the cells, which is precisely measured
by nanoscale thermometry. This allows one to optimize the
PTT conditions by varying concentration of gold nanorods
associated with cells and laser power density. During the
PTT procedure, such an approach enables an accurate
determination of the percentages of apoptotic and necrotic
cells using 2D and 3D models. According to the performed
cell experiments, the inuence of temperature increase
during the PTT on cell death mechanisms has been
veried and determined. Our investigations can improve
the understanding of the PTT mechanisms and increase its
therapeutic eciency while avoiding any side eects.
Keywords: uorescent nanothermometry; laser-induced
heating; necrosis/apoptosis; photodynamic therapy.
1 Introduction
Malignant skin neoplasms, especially melanomas, are
highly aggressive and result in approximately 65% of all
skin cancer deaths and a long-term survival rate of 5% [1].
At the moment, the main therapeutic approaches used
for the treatment of melanoma include chemotherapy,
surgery, immunotherapy, and radiotherapy [2–7]. How-
ever, these methods of treatment are not always satis-
fying due to possible side eects, cell-resistance to the
chemotherapeutic drugs, and radioactive emission [8–11].
As an alternative, photothermal therapy (PTT) can
be considered as an extensively developing method of
cancer treatment [12–15]. This technique requires the use
of light-sensitive nanoscale objects (e.g., biomolecules,
nanoparticles etc.) that can convert light energy into heat.
By applying the laser irradiation to the zone of interest
(e.g., the tumor with impregnated light-responsive agents),
it induces hyperthermia, which leads to the death of
cancerous cells [16–18]. The main advantages of the PTT
Open Access. © 2022 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International
License.

4324 |O. O. Peltek et al.: Fluorescence-based nanothermometry
include spatiotemporal selectivity of treatment, enhanced
immunogenicity, and lack of the protective mechanisms of
cancer cells [19–21].
At present, the number of preclinical and clinical
trials on the use of PTT for melanoma treatment is rapidly
growing. There are already several completed clinical
trials on PTT. For example, NCT00848042 for refractory
and/or recurrent head and neck tumors (2008–2014),
NCT01679470 for metastatic lung tumors (2012– 2014), and
NCT02680535 for localized prostate cancer (2016–2021).
All these clinical trials employed AuroLase Au NPs as
a photosensitive agent for systemic administration [22].
The latest clinical trial on the use of PTT has recently
demonstrated results for the treatment of localized prostate
cancer (NCT02680535). As a result of the PTT, 15 out of
16 patients fully recovered, and a year later, a biopsy
conrmed no relapse. These data speak in favor of the
possibility of using PTT not only for the treatment of
malignant tumors near the skin surface, but also for the
treatment of malignancies in the deeper tissues, using
optical ber for supplying radiation.
In order to improve the eciency of PTT cancer treat-
ment, an understanding of dierent cell death mechanisms
is crucial. The cell death induced by hyperthermia usually
occurs via two possible pathways: necrosis or apoptosis
[23–25]. Apoptosis is referred to as programmed cell death.
In contrast, necrosis is an uncontrolled form of cell death
that is induced by external injuries and in most cases is
caused by membrane rupture. During cancer therapy, the
failure to regulatethe apoptosis/necrosis process can result
in severe adverse eects [26]. During PTT, the cell death
pathway depends on the intracellular temperature induced
by laser heating [27,28]. Thus, it is important to accurately
measure and control the intracellular temperature reached
during the PTT treatment.
There are a number of works devoted to cell thermom-
etry at the nanoscale level. The main approaches of nan-
othermometry mostly utilize organic dyes, quantum dots,
upconverting nanoparticles, and nanodiamonds [29–33].
However, despite the great number of available meth-
ods, to the best of our knowledge, there are no works
that managed to measure the intracellular temperature
distribution during the PTT. The closest works on this
subject describe the eect of PTT based on the temperature
of the cell media that contained gold nanorods (Au NRs),
during laser irradiation using infrared thermometry [23,
34]. However, we believe that dierent approach can be
used for intracellular temperature distribution measure-
ments to obtain temperatures directly within a cell.
To achieve this goal, we decided to apply organic
dye (Rhodamine B, RhB) for uorescent lifetime-based
thermometry due to the simplicity of temperature measure-
ments and easy access to the organic dyes in almost every
chemical laboratory. Au NRs were used as nanoheaters
when performing PTT using a near-infrared laser (NIR,
1064 nm ber laser, 100 kHz). To evaluate the cell
death pattern apoptosis and necrosis assay using ow
cytometry was performed. Additionally, the change in
Bax gene expression was investigated in irradiated cells,
as it encodes apoptosis-inducing protein BCL2L4 and its
expression was elevated in cells subjected to heat-induced
apoptosis [35–37]. The roadmap of the described study is
presented in Figure 1.
We believe the results of this work will enable a
better understanding of the inuence of the intracellular
temperatures reached during the PTT on cell death pattern.
Additionally, the reported ndingscan serve to increase the
eciency of PTT and decrease the adverse eects that may
occur during this procedure. Furthermore, we believe that
the use of methods for intracellular temperature estimation
is not limited only to PTT, but they can nd their application
in other elds of knowledge.
2 Experimental section
2.1 Materials
For Au NRs synthesis: gold (III) chloride trihydrate (HAuCl4·3H2O,
≥99.9%, Sigma-Aldrich), ascorbic acid (AA, ≥99.0%, Sigma-Aldrich),
sodium borohydride (NaBH4, 98%, Sigma-Aldrich), cetyltrimethy-
lammonium bromide (CTAB, ≥99%, Sigma-Aldrich), sodium oleate
(NaOL, >97.0%, Sigma-Aldrich), and amine-poly(ethylene glycol)-
thiol (NH2-PEG-SH, MW 1.000, Laysan Bio, China) were all used
without additional purication.
For cell cultures: Alpha Minimum Essential Medium (Alpha-
MEM) was purchased from Biolot, Russia. Phosphate-buered saline
(PBS), and UltraGlutamine I were purchased from Lonza,Switzerland.
Fetal bovine serum (FBS) was obtained from HyClone, USA. Trypsin-
EDTA solution was purchased from Capricorn Scientic, Germany.
Rhodamine B (RhB, ≥95%) and calcein acetoxymethyl (Calcein AM)
were purchased from Sigma-Aldrich. AlamarBlue cell viability reagent
was purchased from Invitrogen, USA. APC Annexin V apoptosis
detection kit with 7-AAD was purchased from BioLegend, USA.
2.2 Synthesis of functionalized Au NRs
Au NRs were synthesized using seed-mediated growth approach and
binary surfactant mixture according to the protocol developed by Xe
et al. [38] Surface coating of CTAB-stabilized Au NRs was realizedwith
ligand exchange procedure [39]. Details and the full data set are given
in the Supporting Information.

O. O. Peltek et al.: Fluorescence-based nanothermometry |4325
Figure 1: Schematic illustration of roadmap in this work: (i) estimation of temperature dependence of RhB fluorescence lifetime versus the
applied temperature; (ii) temperature estimation of laser-induced heating of RhB; (iii) thermometry of an individual cell with applied external
heating; (iv) measuring of temperatures of 2D cell model under laser-induced heating; (v) evaluation of the percentage ratio of necrotic and
apoptotic cells under laser-induced heating; (vi) evaluation of the percentage ratio of necrotic and apoptotic cells in tumor spheroid under
laser-induced heating.
2.3 Characterization of Au NPs
The absorption spectra for the aqueous solutions of the functionalized
Au NRs were measured in the 10 mm path quartz cuvettes using
spectrophotometer Shimadzu UV-3600 (400–1300 nm interval). Size
distribution of the Au NPs was estimated using the scanning electron
microscopy (SEM,Carl Zeiss Merlin) at an accelerating voltage of 30 kV.
The sample preparation procedure and technical details arepresented
in the Supporting Information.
2.4 Experimental setup for fluorescence lifetime
measurements
To measure the uorescence lifetimes, time-correlated single photon
counting (TCSPC) method was used. The experimental setup is
based on a home-built confocal microscope for uorescence lifetime
measurements with TCSPC. To provide the heating of gold nanorods,
an additional ytterbium laser source (1064 nm ber laser, 100 kHz,
<140 ns FWHM) was used. The detailed data and optical scheme are
described in the Supporting Information.
2.5 Cells
Murine melanoma cell line (B16-F10 cells) was obtained from the
American Type Culture Collection. Cells were cultured in AlphaMEM
supplemented with 10% of vol. FBS and additional 2 mM UltraGlu-
tamine I. The cell culture was maintained in a sterile humidied
atmosphere containing 95% air and 5% CO2at 37 ◦C.
2.6 Toxicity studies
In order to evaluate the toxicity of Au NRs at dierent concentrations,
AlamarBlue assay was performed. The cell viability was analyzed by
measuring absorbances of the media at 570 and 600 nm with UV−vis
spectrophotometer (Thermo Scientic Multiskan GO). The detailed
protocols are presented in the Supporting Information.
2.7 Au NRs uptake
To evaluate uptakeand association of Au NRs with B16-F10, cells were
incubated with Cy-5 labelled Au NRs. Afterwards uptake was visual-
ized using confocal laser scanning microscopy. The data and detailed
experiment description are presented in the Supporting Information.
2.8 External heating of Rhodamine B (RhB) solution
In order to obtain a calibration curve describing the dependence of
uorescence lifetime of RhB on the temperature, 100 μM of aqueous
RhB solution was used. Values of uorescence lifetime were obtained

4326 |O. O. Peltek et al.: Fluorescence-based nanothermometry
using Picoquant Picoharp 300 time-correlated singlephoton counting
system. Heating of RhB solution was realized using a custom made
temperature control system, where the temperature of the solution
was monitored via a thermocouple. The values of the uorescence
lifetimes of RhB were measured at various temperatures.The obtained
data were tted by single exponential t using Matlab software. This
t was later used as a calibration curve to estimate the RhB solution
temperature based on the measured uorescence lifetime values. The
details and the full data set are given in the Supporting Information.
2.9 Laser-induced heating of Au NRs in RhB solution
To evaluate the laser-induced heating of Au NRs, a freshly prepared
100 μM solution of RhB containing Au NRs with a nal concentration
of 20 μg/mL was used. Fluorescence lifetime measurements were
carried out on the custom-made setup. In order to evaluate the
laser-induced heating of Au NRs in RhB solution, it was irradiated
with a pulsed 1064 nm laser with dierent power densities (up to 36.3
kW/cm2). Before each measurement, the solution was continuously
irradiated for 60 s to stabilize the temperature. In order to estimate
the temperatures of the solution during laser treatment, we used
the previously obtained calibration curve. Using this dependence,
we recalculated the measured uorescence lifetimes into their corre-
sponding temperature values. The results and calculations are given
in the Supporting Information.
2.10 External heating of cells
To estimate local cell temperaturesduring external heating, cells were
seeded on confocal cell imaging dishes. Next day, Au NRs were added
to the cells, and the cells were left overnight. On the following day,
the cells were stained with RhB. For this, 10 μL of 1 mM RhB solution
was added to the cell culture medium, so that the nal concentration
of RhB was equal to 10 μM. After 30 min, cells were washed twice
with PBS to remove non-internalized Au NRs and left in PBS supple-
mented with 4% glucose. Fluorescence lifetime measurements were
performed with custom-made setup. Before measurements, cells were
heated up to a certain temperature (32–47 ◦C) with a custom-made
temperature control system to ensure the uniformity of temperature
distribution in the cell imaging dish. The obtained data was tted by
single exponential t using Matlab software. This t was later used as
a calibration curve to estimate mean intracellular temperature based
on the measured uorescence lifetime values. The detailed protocols
and obtained data are presented in the Supporting Information.
2.11 Laser-induced heating of gold nanorods in cells
In order to heat up cells with an NIR-laser, the cells were seeded
into confocal cell imaging dishes with Au NRs at three dierent
nal concentrations of Au NRs in the culture media (20, 40 and
60 μg/mL). On the following day, the cells were stained with RhB.
After 30 min, the cells were washed twice with PBS and left in PBS
supplemented with 4% glucose. Fluorescence lifetime measurements
were performed with the setup described in Section 2.10. During these
measurements, the temperature of the cell medium was kept at 32
◦C. Prior to the measurement, the cells were continuously irradiated
for 60 s to stabilize the temperature. The previously obtained t
was used in these measurements as a calibration curve to estimate
mean intracellular temperature based on the measured uorescence
lifetime values.
2.12 Flow cytometry
The apoptosis/necrosis assay was performed using ow cytometry
(FACS Aria, BD, USA). For this, cells were seeded in 12-well plates at
the amount of 1.0 ×105per well. Next day,Au NRs were added to each
well at dierent concentrations (20 μg/mL to 60 μg/mL). The nal
volume of cell culture medium in each well was 1 mL. Afterwards,
cells were washed twice with PBS and left in PBS supplemented with
4% glucose. Then, each well was irradiated with an NIR-laser with
dierent power densities (up to 43.2 kW/cm2) for 90 s. One hour after
the irradiation, the cells were detached using trypsin-EDTA (V=200
μL per well) solution and stained with 7-AAD and APC Annexin V
according to the protocol provided by the manufacturer (BioLegend,
USA).
2.13 Bax gene expression analysis
The eect of induced hyperthermia on cell apoptosis was further
evaluated by estimating the increase of Bax gene expression using
real-time polymerase chain reaction (PCR). For this, cells were seeded
in 12-well plates at the amount of 1.0 ×105cells per well. Next day, Au
NRs were added to each well at dierent concentrations (20 μg/mL
to 60 μg/mL) and incubated overnight at 37 ◦С,5%CO
2. Afterwards,
cells were washed twice with PBS and left in PBS supplemented with
4% glucose. Then, each well was irradiated with an NIR-laser with
dierent power densities (up to 43.2 kW/cm2) for 90 s. One hour after
the irradiation, total RNA was extracted and puried using an RNA
extraction kit (Evrogen, Russia). Afterwards cDNA was synthesized
from total RNA and real-time PCR was carried out. The detailed
protocols are described in the Supporting Information.
2.14 Formation of tumor spheroid
Spheroids were obtained using the “hanging drop” technique
described by Timmins et al. [40,41]Briey,15μL of culture medium
which contained 5000 B16-F10 cell s were carefully dropped onto the
inside cover of a 35 mm Petri dish. The dish itself was lled with 1.5
mL PBS to prevent evaporation of cell media. The cover of the dish
was placed back on the Petri dish, and the cells were incubated at 37
◦Cand5%CO
2. The cell culture medium inside the drop was changed
each 3 days, and after 6 days, the spheroid formation was complete.
2.15 Laser-induced heating of Au NRs in spheroid
In order to heat the 3D tumor spheroids with a laser, Au NRs were
added to the previously prepared spheroids at a nal concentration
of 60 μg/mL. The next day, spheroids were washed twice with PBS
to remove the non-internalized Au NRs and carefully transferred to a
separate Petri dish prior to the irradiation. Then, each spheroid was
irradiated for 90 s with an NIR-laser with dierent power densities
(up to 14.2 kW/cm2). After the irradiation, each spheroid was stained
with calcein AM, APC Annexin V, and 7-AAD. Cells were incubated
for 15 min at room temperature before the visualization with a CLSM
(Carl Zeiss LSM 710). The detailed protocols and obtained data are
presented in the Supporting Information.

O. O. Peltek et al.: Fluorescence-based nanothermometry |4327
3 Results and discussion
In this study, we investigate the inuence of intracellular
temperature induced by PTT on the apoptosis/necrosis
ratio of melanoma B16-F10 cells, which was determined
by uorescent lifetime-based thermometry during the PTT
procedure. Therefore, we have divided this study into
several steps, including (i) estimation of temperature
dependence of uorescence lifetime; (ii) temperature esti-
mation of laser-induced heating;(iii) thermometry analysis
of cells at applied external heating; (iv) measuring the
temperatures of 2D and 3D cancer cell models under
laser-induced heating; and (v) revealing the percentage
ratio of necrotic and apoptotic cells during laser-induced
heating. The RhB was used for uorescent lifetime-based
thermometry, and Au NRs were applied as nanoheaters for
PTT procedure (Figure 1).
3.1 RhB as nanothermometer
Thermometry at the nanoscale has gained a lot of interest
during recent years as a powerful and versatile tool for bio-
logical applications. One of the thermometry approaches
is using uorescent dyes sensitive to changes in the
temperature as nanothermometers [42]. This property
mainly manifests in molecular rotors (e.g., RhB). The
uorescent properties of RhB depend on the viscosity of
the surrounding media, which, in turn, depends on the
temperature of the media. In the case of RhB, rotations of
the diethylamino groups on the xanthene ring are respon-
sible for the temperature dependence of the uorescence
lifetime and quantum yield [43].
In order to reveal the dependence of uorescence
lifetime on the temperature of the RhB solution, a water
solution of RhB (100 μM) was gradually heated from 25 ◦C
to 50 ◦C with a heating plate, and the uorescence lifetime
values were measured at dierent temperature points. The
uorescence decay in this and all the further experiments
was tted with a single exponent to determine the values
of uorescence lifetime (Figure S4). The obtained values
of uorescence lifetime are in a good agreement with the
previously reported results of 1.74 ns at 20 ◦CbyBoens
et al. [44] and the temperature dependence reported by
Mercadé-Prieto et al. [45]. The results of our experiments
were tted using exponential t, which revealed the
following dependence:
𝜏=2.967 ⋅e−0.0266⋅T(1)
where 𝜏is uorescence lifetime in nanoseconds (ns) and
Tis temperature in Celsius (◦C) (Figure S4).
3.2 Au NRs as heating agents
The Au NRs are extensively used as nanoheaters for
PTT due to their high biocompatibility and superior light
absorbing properties [46]. Therefore, for the purpose of
this study they were chosen as model nanoheaters. The
morphological properties of the synthesized Au NRs were
tailored to ensure that the peak absorbance lies in the
NIR region (1160 nm) to cover the therapeutic window of
biological tissues [47]. The surface of the obtained Au NRs
was additionally modied with H2N-PEG-SH via ligand-
exchange procedure to increase the solubility of these NPs
in aqueous solutions and boost the cell association rate due
to the positively charged surface [48]. The SEM imageswere
obtained to evaluate the morphology of the synthesized
Au NRs (Figure 2A). According to the SEM study, the length
of the obtained Au NRs was 115.5 ±14.8 nm, the width
was 20.1 ±1.9 nm, and thus their aspect ratio is ∼5.75.
The synthesized Au NRs had two absorbance peaks: one at
∼510 nm, which corresponded to transversal plasmon, and
the second at 1160 nm, which corresponded to longitudinal
plasmon (Figure 2B).
Importantly, the presence of plasmonic NPs in the
solution aects the uorescence lifetime of RhB [49]. There-
fore, the previously obtained calibration curve for a simple
RhB solution cannot be used to determine the temperature
of the solution containing the Au NRs. Consequently,
in a manner similar to the previous experiment, a new
dependence of uorescence lifetime on temperature for
asolutionofRhB(100μM) and Au NRs (20 μg/mL) was
found:
𝜏=2.683 ⋅e−0.0253⋅T(2)
ItcanbeseenthatinthepresenceofAuNRs,theuo-
rescence lifetime has decreased signicantly (Figure S4)
due to non-radiative energy transfer between the RhB
molecule and Au NRs. Additionally, it can be noticed that
the increase of the solution temperature diminishes the
dierence in uorescence lifetimes in the presence of Au
NRs.
The obtained dependence then was used to evalu-
ate the temperature change that occurs due to the NIR
laser irradiation of Au NRs within RhB solution. For the
following experiments a pulsed NIR laser with a wave-
length of 1064 nm was used (Figure 2C). The uorescence
lifetimes were measured at various laser power densities
(up to 36.3 kW/cm2). Several studies have shown that
pulse laser in combination with Au NPs can cause cell
death not only due to the increased temperature, but
also due to explosion of the bubbles generated on the
surface of plasmon nanoparticles. However, in our work

4328 |O. O. Peltek et al.: Fluorescence-based nanothermometry
Figure 2: Au NRs characterization. (A) SEM images of AuNRs; (B) absorbance spectrum of Au NRs; (C) scheme of the experiment for
fluorescence lifetime-based thermometry of RhB solution containing Au NRs; (D) the mean fluorescence lifetime of RhB solution in the
presence of AuNRs (40 μg/mL) during NIR laser irradiation and the corresponding calculated temperature values.
the laser power densities are not high enough to induce
this eect described elsewhere [50,51]. Prior to each
measurement, the solution was continuously irradiated
for 60 s to stabilize the temperature. The light energy
distribution within the laser spot follows Gaussian dis-
tribution; therefore, this was taken into account when
determining laser power densities. Additionally, irradiated
cells were precisely placed in the middle of the laser spot.
The measured uorescence lifetimes were recalculated
into temperature values, using the previously obtained
dependence (Eq. (2)) of RhB uorescence lifetime on the
temperature of the solution in the presence of Au NRs.
According to the obtained data, the solution was gradually
heated from the initial room temperature (23 ◦C) up to 52 ◦C
(Figure 2D).
3.3 Intracellular temperature mapping
The uorescence lifetime of the uorophores depends on
the properties of the surrounding media, which can be
used to determine the intracellular temperature. However,
the change in viscosity, pH and hydrophobicity of the
surrounding media also aects the uorescence lifetimes
[43]. This means that the calibration curve for temperature
estimation that we have derived previously for the water
solution of RhB cannot be applied to measure and cal-
culate intracellular temperature. The RhB mainly stains
cell membranes, since it is highly soluble in them, and
thus, the uorescence lifetime increases compared to the
uorescence lifetime of RhB in water. Therefore, a new
calibration curve for RhB-stained cells was necessary for
further experiments.
The stained adherent B16-F10 cells were placed onto
the heating plate, which was positioned on a piezo stage.
Then the cell media was heated up to a certain temperature
(from 32 ◦Cto46◦C), and the uorescence lifetime mea-
surements were performed. The detection rate was limited
to 5% of the excitation repetition rate at the brightest pixel
in order to avoid photon pile-up and prevent falsely shorter
lifetimes. In each measurement, a single cell was scanned

O. O. Peltek et al.: Fluorescence-based nanothermometry |4329
(step was equal to 1 μm) in order to accurately determine
uorescence lifetime distribution within a single cell at
a certain temperature (Figure S7). Finally, the values
were averaged and plotted against the temperature of the
solution (Figure S8). It can be seen that the RhB that stained
cells demonstrates longer uorescence lifetimes compared
to RhB water solution at the same temperatures. As it was
mentioned previously, this eect can be explained by RhB
exhibiting longer lifetimes in more viscous media, such as
cell membranes [43].
Then the obtained data were tted using the expo-
nential t, revealing the following relation between the
uorescence lifetime and intracellular temperature:
T=61.02 ⋅e−0.344⋅𝜏(3)
This equation was further used to determine the
intracellular temperature in the latter experiments. It is
important to underline that unlike the experiment with
RhB water solution, the presence of internalized Au NRs
inside of the cells did not aect the measured uorescence
lifetime values. Furthermore, this technique overall does
not depend on the concentrations of the RhB used for cell
staining. We have compared various concentrations of RhB
(from 10 μMto50μM) and found no clear dependence of
uorescent lifetime values on the concentration of staining
solution (Figure S9). Furthermore, since the dye was absent
in the surrounding media and only cells were stained
with RhB, uorescence lifetimes and therefore intracel-
lular temperatures were measured and not extracellular
temperatures of the surrounding medium.
To perform further experiments, it was necessary to
choose appropriate concentrations of Au NRs. For this,
the toxicity of Au NRs (from 1 μg/mL up to 60 μg/mL)
was evaluated on murine melanoma cells B16-F10, and
it was shown that none of these concentrations has
cytotoxic eect (Figure S5). Thus, three concentrations
were chosen for the further experiments: 20, 40 and
60 μg/mL. Furthermore, the cell uptake of Au NRs was
evaluated using CLSM (Figure S6). It can be seen that
some Au NRs are localized within the cells (presumably
in lysosomes), forming aggregates. Another part of Au NRs
is distributed on the surface of the cell membrane. Thus, it
can be assumed that such a distribution may further lead to
a uniform heating of a tumor cell. After 24 h of incubation,
the Au NRs were added to the cells. On the following
day, the cells were stained with 10 μMsolutionofRhB
and placed on the heating plate in the uorescent lifetime
measuring setup. The temperature of the cell culture media
was kept at 32 ◦C during the whole experiment. Prior to the
uorescence lifetime measurements of RhB-stained cells,
they were irradiated with the NIR laser for 60 s to achieve
uniform heating. After each successful scan, the power
density was increased and a new uorescence lifetime
measurement was performed. Every uorescence lifetime
measurement yielded a uorescence lifetime distribution
within a single chosen cell for a specic laser power density
(Figures 3A,S10, S11). The diameter of the laser beam
(2 mm) was larger than the size of cells to ensure the even
distribution of the laser power density. Afterwards, the
values of the uorescence lifetime within each cell were
averaged and plotted against the laser power density. It can
be seen that higher concentrations of Au NRs present in the
cell culture medium lead to lower uorescence lifetimes
achieved during laser irradiation with the same power
density and, therefore, higher intracellular temperatures
(Figure 3B and C).
The measured average uorescence lifetimes were
converted into intracellular temperature values using the
previously obtained calibration curve (Figure S8). It can
be seen that the intracellular temperatures in the case
of the lowest Au NRs concentration (20 μg/mL) do not
exceed 36–37 ◦C even at the highest power densities,
therefore, this concentration was deemed too low for
further therapeutic use. Nonetheless, the intracellular
temperatures during the irradiation with a power density
equal to 42.9 kW/cm2in the case of the highest Au NRs
concentration (60 μg/mL) were as high as 46.3 ±2.9 ◦C,
which is enough to induce apoptosis in most of the
treated cancer cells according to the published works [16].
Although the chosen method for temperature measure-
ments may lack the accuracy compared to other works
[32,52–54], it demonstrates precision comparable to other
uorescence-based approaches. Furthermore, it can be
used to estimate temperature distribution within the whole
cell during the laser irradiation unlike nanoparticle-based
measurements.
3.4 Apoptosis versus necrosis in 2D cell
model
To evaluate the inuence of the intracellular temperatures
reached during laser irradiation of internalized Au NRs
on the type of cell death, ow cytometry with Annexin V
and 7AAD staining was performed. The goal of this exper-
iment was to determine the intracellular temperatures
suciently high to induce apoptosis in most of the cancer
cells and to nd the optimal concentrations of Au NRs,
as well as corresponding laser power densities required to
achieve them.
For this, cells were incubated overnight with three
dierent Au NRs concentrations (20, 40 and 60 μg/mL).

4330 |O. O. Peltek et al.: Fluorescence-based nanothermometry
Figure 3: Fluorescence-based thermometry of B16-F10 cells. (A) Fluorescence lifetime images of B16-F10 cells incubated with AuNRs (40
μg/mL) and stained with RhB. Images were obtained while the cell was irradiated with an NIR laser with a specific power density. The image
contains fluorescence intensity map, fluorescence lifetime map and the fluorescence lifetimes presented in histogram form. Scale bar
corresponds to 5 μm; (B) average intracellular fluorescence lifetime of RhB depending on laser power density in the cells incubated with
different concentrations of AuNRs (20, 40 and 60 μg/mL); (C) average intracellular temperatures depending on laser power density in the
cells incubated with various concentrations of Au NRs. The data represent the mean and standard deviation of three independent samples.
Afterwards, each plate well with cells was irradiated
with a certain laser power density (up to 43.2 kw/cm2)
for 90 s. One hour after the irradiation, the cells were
stained with Annexin V and 7-AAD and analyzed using
ow cytometry. The plasma membrane of intact cells is
composed of lipids that are asymmetrically distributed
on the inner and outer leaet of the membrane, with
phosphatidylserine normally restricted to the inner leaet
and exposed during apoptosis [55]. Marking the phos-
phatidylserine with Annexin V allows distinguishing the
apoptotic cells from the necrotic cells with permeable
membranes that fail to exclude the 7-AAD Therefore, the
results obtained from ow cytometry were interpreted in
the following manner: cells negative in both channels
(AnnV−/7-AAD−) were considered to be live, Annexin
V positive and 7-AAD negative (AnnV+/7-AAD−) cells
were considered to be in early apoptosis, cells positive
in both channels (AnnV+/7-AAD+) – secondary necrotic
(late apoptotic cells that suer from the loss of membrane
integrity in the absence of phagocytic cells) and, nally,
Annexin V negative and 7-AAD positive (AnnV−/7-AAD+)
cells were identied as necrotic (Figure 4B)[34].
From the obtained data, it can be seen that the
amount of the apoptotic cells increases both with the
increase of concentration of Au NRs added to the cells
and the laser power density. At the power density equal to
43.2 kW/cm2for both 40 and 60 μg/mL of Au NRs, more
than 80% of all the cells were apoptotic. Additionally,
it is interesting to notice that for 20 μg/mL of Au NRs;
even the highest laser power density was not enough to
induce signicant cell death. However, if we take into
account the data from the previous experiment, it becomes
evident that for the case of 20 μg/mL of added Au NRs, the
intracellular temperature did not exceed 36–37 ◦C, which
explains why no apoptotic cells were detected. At the same
time, at the highest laser power density (43.2 kW/cm2),
the cells that were incubated with 40 and 60 μg/mL of
Au NRs demonstrated intracellular temperatures equal to
43.1 ±3.1 ◦Cand46.3±2.9 ◦C, respectively. This tempera-
ture was suciently high to induce apoptosis in most of the

O. O. Peltek et al.: Fluorescence-based nanothermometry |4331
Figure 4: Viability of B16-F10 cells and Bax gene expression after laser irradiation. (A) The percentages of AnnV+, AnnV−, and 7-AAD+cells
incubated with different concentrations of Au NRs (20, 40, and 60 μg/mL) after irradiation with various laser power densities. (B, C) the flow
cytometry data of Annexin V and 7AAD stained B16-F10 cells incubated with 40 μg/mL of Au NRs after no irradiation (B) and after irradiation
with power density equal to 36.5 kW/cm2(C). (D) Effect of Au NRs and laser irradiation on the expression level of Bax gene. In these
experiments GAPDH was chosen as the housekeeping gene. The change in gene expression is shown as fold changes referring to untreated
control cells. The data represent the mean and standard deviation of three independent samples.
cell population, with a varying number of cells undergoing
secondary necrosis.
If we compare this data to the results of intracellular
measurements, it becomes evident that signicant cell
apoptosis occurs once the temperatures reach more than
42 ◦C. As we increase the temperature further, the per-
centage of secondary necrotic cells increases up to 50%
(60 μg/mL of Au NRs at 43.2 kW/cm2). Nonetheless, it is
worth mentioning that the temperatures reached during
PTT in all the samples were not high enough for primary
necrosis to exceed more than 5.5% of the cells.
The cell apoptosis can be additionally determined
by analyzing the expression level of the Bax gene in
the cells using real time PCR. The Bax gene encodes
BCL2L4 protein that induces cell death by altering cell
mitochondria [56]. Various works have demonstrated the
increase of expression level of Bax gene after heat-induced
apoptosis [35–37]. In our case it can be seen that the
expression of Bax gene was not increased in the cells
that were treated with laser power densities below 26.7
kW/cm2(Figure 4D). These results are in good agreement
with the data from ow cytometry. As the intracellular
temperature during laser irradiation begins to exceed 42 ◦C
(40 μg/mL and 60 μg/mL of Au NRs at 26.7 kW/cm2)theBax
expression increases more than 15-fold (Figure 4D). This is
reected by the high percentage of apoptotic cells detected
using ow cytometry in the samples treated with the same
conditions. Interestingly, the Bax expression signicantly
decreases in the cells incubated with 60 μg/mL of Au NRs
and treated with the power density equal to 43.2 kW/cm2.
In this case the intracellular temperature exceeds 47 ◦C,
which presumably leads to cell necrosis, thus, resulting in
lower Bax gene expression levels.
3.5 Apoptosis versus necrosis in 3D cell
model
In order to further investigate the inuence of laser-
induced Au NRs-mediated PTT, the following experiments
were performed on a 3D spheroid tumor model of B16-
F10 cells. Tumor spheroids are commonly used in drug
development since they can replicate physiological cell
environment to a higher extent compared to the usual 2D
cell culture [41,57,58]. When it comes to PTT, the use of
the spheroid tumor model can oer a new insight into the
optimal laser power densities and Au NRs concentrations.

4332 |O. O. Peltek et al.: Fluorescence-based nanothermometry
The tumor spheroids were incubated overnight with
60 μg/mL of Au NRs and afterwards subjected to PTT for
90 s, with laser power densities indicated above. Then,
the tumor spheroids were stained with Calcein AM, APC
Annexin V and 7-AAD. The live, apoptotic and necrotic
cells were visualized using the CLSM (Figure 5A and B).
At laser power density equal to 8.1 kW/cm2,acertain
amount of apoptotic cells on the periphery of the spheroid
can be noticed with only a few 7-AAD+cells. As power
density was increased up to 9.2 kW/cm2, the number of
Calcein AM positive cells dropped signicantly, and the
number of Annexin V positive cells greatly increased. Since
the number of 7-AAD+cells is insignicant, this means
that almost all the cells died via apoptosis. Finally, at
14.2 kW/cm2, a large portion of spheroid cells became
7-AAD+and, therefore, were considered to be necrotic.
Overall, it can be seen that laser power densities required
to induce a signicant apoptosis in tumor spheroid were
much lower compared to those required for 2D culture.
This can be explained in the following manner: the
tumor spheroid has decreased the heat exchange rate
with the surrounding media compared to the 2D cell
culture, and thus, during PTT, the same Au NRs concen-
trations and laser power densities may result in much
higher intracellular temperatures. However, these temper-
atures could not be identied due to inherent limitations
of the optical setup. The suggested uorescence-based
thermometry was developed to measure intracellular
temperatures at nanoscale of 2D and 3D cell models,
thus it cannot be utilized for temperature measurements
of larger objects without signicant alterations in the
technique.
Figure 5: PTT of tumor spheroids. (A) The
CLSM image of the tumor spheroid incubated
with AuNRs (60 μg/ml) after PTT for various
laser power densities. The live cells were
stained with calcein AM (green), apoptotic
cells – with APC Annexin V, and necrotic
cells – with 7-AAD. Scale bar is equal to 5 μm.
(B) Relative intensities of the corresponding
fluorescence values calculated from the
obtained CLSM images. The data represent
the mean and standard deviation of three
independent samples.

O. O. Peltek et al.: Fluorescence-based nanothermometry |4333
4 Conclusions
In this work, we have utilized a new approach for the
evaluation of intracellular temperatures under the condi-
tions similar to those of PTT. The developed thermometry
method allowed us to evaluate the change of intracellular
temperature due to laser-induced heating of internalized
Au NRs. We have demonstrated that intracellular tem-
perature above 42 ◦C was required to induce apoptosis
in more than 80% of B16-F10 melanoma cells. At the
same time, as intracellular temperature continues to rise, a
greater number of cells undergo secondary necrosis. Over-
all, the developed thermometry approach allows precise
estimation of the intracellular temperature during PTT,
which, to the best of our knowledge, has not been reported
elsewhere. These ndings help to better understand how
the temperature changes inside the cell duringPT T and can
be used in the future to further optimize the parameters for
the development of novel PTT-based methods of cancer
treatment.
Author contribution: All the authors have accepted respon-
sibility for the entire content of this submitted manuscript
and approved submission.
Research funding: This work was supported by the Russian
Science Foundation (Project 21-72-30018). Part of this work
related to the interaction of NPs with cells and spheroids
was supported by the Russian Science Foundation (Project
21-75-10044). Part of this work related to the synthesis of
Au NPs was supported by the Russian Science Foundation
(Project 22-75-10011).
Conflict of interest statement: The authors declare no
conicts of interest regarding this article.
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Supplementary Material: The online version of this article offers sup-
plementary material (https://doi.org/10.1515/nanoph-2022-0314).