Lasers in Medical Science (2004) 18: 204–206
DOI 10.1007/s10103-003-0281-7
O R I GI N A L A R T IC L E
R. Bortoletto Æ N. S. Silva Æ R. A. Zângaro
M. T. T. Pacheco Æ R. A. Da Matta
C. Pacheco-Soares
Mitochondrial membrane potential after low-power laser irradiation
Received: 24 August 2003 / Accepted: 21 October 2003 / Published online: 14 January 2004

Springer-Verlag London Limited 2004
Abstract We used the lipophilic cationic fluorescent dye
5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethyl-benzimidazol-
carbocyanine iodide (JC-1) to determine mitochondrial
membrane potential (mDw) in Hep-2 cells after irradia-
tion with low-power laser (k=635 nm). Through this
methodology it was possible to analyze the variation on
mitochondrial number and mDw, in cells irradiated for
100, 150 and 200 s with energy density of 100 mJ/cm2.
Our results show that JC-1 dye allows the identification
of populations with different mitochondria morphology
as well as the functionality of this organelle in the cells
incubated for 1, 6 and 24 h, after irradiation with low-
power laser.
Keywords Hep-2 cells Æ Laser Æ Mitochondrial
membrane potential
Introduction
Mitochondrial membrane potential (mDw) is directly
related to ATP production. Considering the role that the
mitochondria have in many pathophysiological condi-
tions, such as calcium homeostasis, oxygen radical
generation and control of apoptosis [1, 2], assessment of
R. Bortoletto Æ N. S. Silva Æ R. A. Zângaro
M. T. T. Pacheco Æ C. Pacheco-Soares (&)
Laboratório de Biologia Celular e Tecidual,
Instituto de Pesquisa e Desenvolvimento,
Universidade do Vale do Paraı́ba,
Av. Shishima Hifumi 2911,
12244-000 São José dos Campos, S.P., Brazil
E-mail: cpsoares@univap.br
Tel.: +55-12-39471143
Fax: +55-12-39471149
R. A. Da Matta
Centro de Biociências e Biotecnologia,
Universidade Estadual do Norte Fluminense,
Av. Alberto Lamego 2000,
28015-620 Campos dos Goytacazes, R.J., Brazil
mDw in cells is a growing interest. The effects of laser
light on biological systems have been widely investigated
in vivo and in vitro [3]. Several studies demonstrated
that mitochondria are sensitive to visible monochro-
matic light irradiation [3]. Mitochondria irradiated with
light in the red region presents higher mDw and proton
gradient, leading to changes in mitochondrial optical
properties, modifying some NADH-linked dehydroge-
nase reactions, and increasing the exchange rate of
ADP/ATP, as well as RNA and protein synthesis [4].
The irradiation of isolated mitochondria also induces
changes in mitochondrial transcription and translation,
increasing a cascade of reactions and a number of the
components of the respiratory chain (e.g., cytochromes,
cytochrome oxidase and flavine dehydrogenase) [5, 6].
The aim of this study was to examine mDw of a cell line
using a fluorescent dye, the lipophilic cationic probe
5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢,-tetraethyl-benzimidazol-
carbocyanine iodide (JC-1), after low-power irradiation.
This dye is advantageous over other potential-sensitive
ones, such as rhodamines and other carbocyanines,
because a color change from green to yellow indicates
higher membrane potential [7].
Materials and methods
The human Hep-2 (trachea carcinoma) cell line was routinely cul-
tured using Minimum Essential Medium (MEM, Gibco) supple-
mented with 10% fetal bovine serum, penicillin (100 U/ml),
streptomycin (100 mM/ml) and fungizone (0.25 lg/ml). Cells were
cultured at 37C in a 5% CO2 atmosphere. For the experiments,
cells were cultured on Petri dishes containing cover slips until a
monolayer was formed.
Hep-2 cells were irradiated for 100, 150 and 200 s with a gal-
lium aluminum arsenide (GaAlAs) diode laser—Thera Lase
(k=635 nm, 10 mJ/cm2). The laser beam was conducted through a
16-mm-diameter optical fiber directly to the Petri dish containing
the cells. After this, cells were further cultivated for 1, 6 or 24 h,
stained for 10 min with JC-1 Molecular Probes Inc. (Eugene, OR)
(10 lM) in MEM, washed in PBS and fixed for 10 min with 4%
freshly prepared formaldehyde in 0.1 M phosphate buffer pH 7.2.
Cover slips containing cells were washed in PBS and mounted with
N-propyl-gallate, and mDw was analyzed and photographed using

Fig. 1 Non-irradiated Hep-2 cells, cultured for 1 h (arrow), stained
with JC-1. Cells present mitochondria with filamentous aspect
(arrowhead) and a low membrane potential. Bar 10 lm
Fig. 2 Hep-2 cells were irradiated with diode laser for 150 s and
stained with JC-1 1 h after irradiation. Mitochondria present a
granular aspect (arrow), with high membrane potential, observed
by orange green fluorescence (arrowhead). Bar 10 lm
Fig. 3 Hep-2 cells were irradiated with diode laser for 150 s and
stained with JC-1 24 h after irradiation. The cells present
mitochondria with filamentous aspect (arrow). A low membrane
potential (green fluorescence labeling) similar to the control is
observed (arrowhead). Bar 10 lm
a Leica DMLB microscope equipped with epi-fluorescence, a HBO
100 W mercury lamp and the corresponding filter sets for fluores-
cence microscopy: blue—FITC excitation filter 450 to 490 nm;
205
barrier 510 nm; emission 520 to 560 nm. Cell fixation caused col-
oration alteration of JC-1 aggregates, turning the red emission of
live cells into yellow, which indicates higher mDw. Green emission
indicates low mDw. Experiments were performed 12 times in
triplicate (cover slips) and representative images are shown.
Results
Non-irradiated cells (control) labeled with JC-1 pre-
sented mitochondria with filamentous aspect and low
membrane potential (green fluorescence labeling) in all
periods tested (1, 6 and 24 h) (Fig. 1). Irradiated cells
that were cultured for 1 and 6 h presented a similar
intensity of the mDw (yellow fluorescence labeling)
indicating high ATP synthesis (Fig. 2). However, irra-
diated cells cultured for 6 h presented their mitochon-
dria with a granular aspect (Fig. 2). No difference was
observed between the cells irradiated for 100, 150 and
200 s.
Cells irradiated for 100 s that were cultured for 24 h
post-irradiation presented mitochondria with a fila-
mentous aspect as observed in control cells. However,
cells irradiated for 150 and 200 s present a low mem-
brane potential and mitochondria with granular aspect
(Fig. 3).
Discussion
Our results demonstrated that the probe JC-1 is a good
marker to evaluate the action of low-power laser in the
mDw. It was observed that the fluorescence intensity for
all the irradiated cases was larger than non-irradiated
cells, indicating an increase in ATP synthesis. These re-
sults are in accordance with previously published in vitro
studies [8, 9, 10]. The only exception observed was that
after 24 h of culture post-irradiation with 150 and 200 s,
cells presented a membrane potential similar to the
control. This may be explained by the consumption of
the extra energy induced by the radiation laser. Twenty-
four hours after incubation the cells irradiated in the
times of 100, 150 and 200 s present a low membrane
potential, similar to the control, indicating that they
need a long period to consume the energy supplied by
the radiation laser.
Even though several works have demonstrated the
effect of low-power laser irradiation in the increase of
the mitochondria membrane potential and proton gra-
dient, other studies are necessary to analyze the ultra-
structural modifications produced in cells irradiated with
red light.
Acknowledgments This work was supported by grants from FA-
PESP, UNIVAP- Universidade do Vale do Paraı́ba, FAPERJ and
CNPq.
206
References
1. Korshunov SS, Skulachev VP, Starkov AA (1997) High pro-
tonic potential actuates a mechanism of production of reactive
oxygen species in mitochondria. FEBS 416:15–18
2. Heiskanen KM, Bhat MB, Wang HW, Ma J, Nieminen AL
(1999) Mitochondrial depolarization accompanies cytochrome c
release during apoptosis in PC6 cells. J Biol Chem 274:5654–5658
3. Karu TI, Pyatibrat L, Kalendo G (1995) Irradiation with
He-Ne laser increases ATP level in cells cultivated in vitro.
J Photochem Photobiol B 27:219–223
4. Abergel RP, Meeker CA, Lam TS, Dwyer RM, Lesavoy MA,
Uitto J (1984) Control of connective tissue metabolism by
lasers: recent development and future prospects. J Am Acad
Dermatol 11:1142–1150
5. Kato M, Shinzawa K, Yoshikawa S (1981) Cytochrome
oxidase is a possible photoreceptor in mitochondria. Photo-
biochem Photobiophys 2:263–269
6. Gordon SA, Surrey K (1960) Red and far-red light action on
oxidative phosphorylation. Radiat Res 12:325–339
7. Reers M, Smith TW, Chen LB (1991) J-aggregate formation of
a carbocyanine as a quantitative fluorescent indicator of
membrane potential. Biochem 30:4480–4486
8. Karu TI (2000) Mechanisms of low-power laser light action on
cellular level. In: Z. Simunovic (ed) Lasers in medicine and
dentistry. Vitgraf, Rijeka, pp 97–125
9. Karu T (1999) Primary and secundary mechanisms of action of
visible to near-IR radiation on cells. J Photochem Photobiol B
49:1–17
10. Hilf R, Murant RS, Narayanan U, Gibson SL (1986) Rela-
tionship of mitochondrial function and cellular adenosine tri-
phosphate levels to hematoporphyrin derivate-induced
photosensitization in R3230AC mammary tumors. Cancer Res
46:211–217