Gen. Physiol. Biophys. (2005), 24, 209—220
Mitochondrial Alterations Induced
by 532 nm Laser Irradiation
P. Kaššák1, T. Przygodzki2, D. Habodászová1, M. Bryszewska2
and L. Šikurová1
1Division of Biomedical Physics, Faculty of Mathematics, Physics and Informatics,
Comenius University, Bratislava, Slovakia
2Department of General Biophysics, University of Lodz, Lodz, Poland
Abstract. Mitochondrial alterations were monitored after low power green laser
(532 nm, 30 mW) irradiation in the case of whole cells (B-14) and isolated mitochon-
dria (from Wistar rat heart). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide (MTT) assay products were significantly higher (by 8%) in irradiated
B-14 cells as compared to non-irradiated controls. Mitochondrial transmembrane
potential of B-14 cells, measured by means of a fluorescent probe 3,3’-dihexyl-
oxacarbocyanine iodide (DiOC6(3)), significantly increased (by 13%) after expo-
sure to green laser irradiation. Another MTT assay was used for isolated mito-
chondria suspensions in order to examine the effect of green laser irradiation on
stimulation of processes related to oxidative phosphorylation. It revealed 31.3%-
increase in MTT assay products in irradiated mitochondria as compared to controls.
Laser irradiation of isolated mitochondria suspension did not significantly change
1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, indicating that mito-
chondrial membrane fluidity was not affected by laser light. Fluorescence emission
spectra of irradiated as well as non-irradiated mitochondria suspensions showed
fluorescence maximum at 635 nm, corresponding to emission of Protoporphyrin
IX, which was significantly lower (by 20.7%) in irradiated sample.
Key words: Mitochondria — Transmembrane potential — Oxidative phosphory-
lation — Membrane fluidity — Laser irradiation — Protoporphyrin IX
The first publication about low-level laser therapy (LLLT) appeared more than 30
years ago (Kovács et al. 1974). Since then, the effectiveness and applicability of
a variety of light sources, in the treatment of a wide range of medical conditions
Correspondence to: Peter Kaššák, Division of Biomedical Physics, Faculty of Mathe-
matics, Physics and Informatics, Comenius University, Mlynská Dolina F1, 842 48 Brati-
slava 4, Slovakia. E-mail: email@example.com
Kaššák et al.
has thoroughly been investigated, in vitro as well as in vivo (for reviews see Conlan
et al. 1996).
In LLLT, the question is no longer whether light has biological effects but
rather how radiation from therapeutic lasers and light emitting diodes (LEDs)
works at the cellular and organism levels and what the optimal light parameters
are for different uses of these light sources with various wavelengths (Karu 2003).
For the lower wavelength bands, there is lack of information about their effect
on biological tissues and cells. Up today, green wavelengths band was not officially
included into LLLT and stands on the edge of research interest in spite of its possible
beneficial use. The fact that green light has small penetration depth (Šikurová et al.
2003) in the biological tissues does not have to be disadvantage as often mentioned,
but also may be used in the very focused and gentle therapeutical interventions.
Wavelengths of 514 and 532 nm have been reported to change heart beating
frequency, activation in contractibility and electrical activity in rat myocardial cells
(Berns et al. 1972; Salet et al. 1979). Furthermore, using LED of wavelength 570 nm,
Vinck et al. (2003) observed stimulation of the chicken fibroblast proliferation. Karu
(1999, 2003) marginally reported in her works absorption of green band light by
fibroblasts and small DNA synthesis activation. Increased activity of the membrane
bound enzyme Na+,K+-ATPase after the irradiation by 532 nm laser light has been
shown (author’s data, not published).
Large number of studies (Conlan et al. 1996; El Batanouny et al. 2002; Webb
and Dyson 2003) has been performed using red or infra-red lasers to observe bios-
timulating effect of these wavelengths on various tissues and cell related processes.
Selected from those, Passarella et al. (1984) showed that irradiation of He-Ne laser
(632.8 nm, 15 mW) generates an extra electrochemical potential and an increase in
ATP synthesis within mitochondria. Later, Karu (2003) suggested that photorecep-
tors might be components of the respiratory chain. Also proposed that in wounds
exhibiting delayed healing, the effect of low intensity visible light (632.8 and 760
nm) might stimulate cells to increase proliferation. Photoreception occurring at the
mitochondrial level may intensify respiratory metabolism and the electrophysiolog-
ical properties of the membrane, thus leading to changes in cell physiology.
In the present work we investigated green laser effect on B-14 cells and iso-
lated rat heart mitochondria in the matter of altered redox state and changes in
membrane properties as well as possible damaging photodynamic effect of the pro-
toporphyrin IX (PpIX) – precursor of heme, located in the mitochondrial membrane
and absorbing light in the range of our stimulation.
Materials and Methods
B-14 cells preparation and culture procedures
Cultures of B-14 (Chinese hamster ovarian) cells were maintained in culture me-
dium (Dulbecco’s modified Eagle’s medium (DMEM) enriched by 10% newborn
calf serum and 1% ampicilin/streptomycin). Cells were plated on 96-well plates
Green Laser Effect on the Mitochondria
in density of 40,000 cells per well approximately 24 h before the experiment and
stored at 37◦C in a humid atmosphere.
Heart mitochondria isolation
Isolation was performed on adult male Wistar rats weighing 290 ± 20 g fed with a
standard Larsen diet and water ad libitum. The animals were sacrificed by decapi-
tation in accordance with the Guide for the Care and Use of Laboratory Animals.
Hearts were placed in an ice-cold isolation solution containing 180 mmol/l KCl,
4 mmol/l EDTA – Tris (pH 7.4) and cut into pieces. Isolation solution enriched by
0.1% bovine serum albumin (BSA) was added together with protease (2.5 mg per g
of heart tissue). Homogenisation with teflon pestle followed and heart mitochondria
were isolated by differential centrifugation (Lehninger et al. 1967). Mitochondria
obtained by this isolation procedure maintained mainly their inner membrane fully
functional but were oriented inside out (Vrbjar et al. 1984; Ziegelhöffer et al. 2003;
Ziegelhöffer-Mihalovičová et al. 2003; Waczulíková et al. 2004). All isolation pro-
cedures were carried out on ice.
Protein content in the isolated mitochondria was estimated by mean of the
method described by Lowry et al. (1951) using BSA as standard. Pellet of isolated
mitochondria was diluted by isolation solution to obtain concentration 0.25 g/ml
In our study, Nd:YAG laser (Raise Electronics, Taiwan) with output power 30 mW
and emitting wavelength of 532 nm was used as a source of polarized and coherent
light. Time of irradiation was set to 20 min obtaining light energy of 36 J. Each
examined sample was irradiated alone receiving the radiant energy per unit area of
the sample surface (fluence) of 1146 J/cm2. Controls were maintained in dark and
at the same conditions as irradiated samples. Irradiation of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium-bromide (MTT; Sigma-Aldrich Inc., USA) solution
itself did not show any changes or production of formazan crystals, respectively.
Trypan blue assay
Trypan blue dye exclusion assay is the most commonly used and accepted method
for the measurement of cell viability. It relies on the alteration in membrane in-
tegrity as determined by the uptake of dye by dead cells, thereby giving a direct
measure of cell viability. We added of 0.4% (w/v) Trypan blue (Sigma-Aldrich Inc.,
USA) to cell suspension after irradiation procedure, mixed well, and scored under
a phase contrast microscope (Li et al. 2003).
This assay is a quantitative colorimetric method to determine cell viability. It
utilizes the yellow tetrazolium salt (MTT) which is metabolized by mitochondrial
dehydrogenase enzyme from viable cells to yield a purple formazan reaction product
Kaššák et al.
(Mosmann 1983). In our experiments, MTT assay was applied in cultured B-14 cells
on wells as well as for rat heart mitochondria suspension in tubes.
On wells: culture medium was replaced with 50 µl of 0.2 mg/ml MTT solution
in the incubation buffer (140 mmol/l NaCl, 5.4 mmol/l KCl, 1.8 mmol/l CaCl2, 0.8
mmol/l MgSO4, 0.9 mmol/l NaH2PO4, 3.8 g/l glucose, 1 mmol/l sodium pyruvate,
20 mmol/l HEPES, pH 7.4).
In tubes: mitochondria suspension was mixed with MTT yielding the final
concentration of MTT 0.2 mg/ml and final concentration of mitochondria 12.5 g/l
in the sample.
In both assays, incubation of cells loaded with MTT lasted two hours. Finally,
solution over the attached or softly spun down cells was gently removed and pro-
duced formazan crystals were dissolved in 50 µl (in wells) and 200 µl (in tube) of
DMSO respectively. Absorbance of dissolved formazan crystals was measured at
560 nm by using Stat Fax-2100 multiwell plate reader (Awareness Technology Inc.,
Florida, USA) and spectrometer Specord M40 (Carl Zeiss, Germany) respectively.
Mitochondrial transmembrane potential measurements
Changes in mitochondrial transmembrane potential of B-14 cells were monitored
using the fluorescent probe 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3); Molec-
ular Probes Inc., USA). Probe solution in final concentration of 50 nmol/l was
freshly prepared in incubation buffer before each measurement from 20 µmol/l
DiOC6(3)/DMSO stock solution. 50 µl of the probe solution was then added to
an appropriate well. Fluoresecence intensity at 535 nm (excitation at 485 nm) was
measured on a Fluoroskan Ascent FL (Labsystems, Finland) multiwell fluorescence
Membrane fluidity measurements
Steady-state fluorescence anisotropy (r) of the fluorescent probe 1,6-diphenyl-1,3,5-
hexatriene (DPH; Sigma-Aldrich, Inc., USA) was evaluated as an indicator of
structural ordering in the hydrophobic core of the membrane, which is in inverse
proportion to membrane fluidity. The stock solution of DPH (5 × 10−4mol/l in
acetone) was further diluted with Tris-KCl-EDTA solution (pH 7.4) to concen-
tration of 2 × 10−6mol/l. Mitochondria suspensions were then stained with the
DPH probe by 1:1 dilution yielding the final DPH concentration of 1×10−6mol/l
and the final concentration of mitochondria of 3.125 g/l. Steady-state fluorescence
anisotropy values were obtained from the Perkin-Elmer LS 45 fluorimeter, based
on measurements of IVVand IVHi.e., the fluorescence intensities polarized parallel
(V) and perpendicular (H) to the vertical plane of polarization of the excitation
beam, respectively. The fluorescence anisotropy is defined by the equation
r = (IVV− GIVH)/(IVV+ 2GIVH)
where G equals IHV/IHHand is the correction factor for instrumental artefacts.
Green Laser Effect on the Mitochondria
Simultaneously, fluorescence emission spectra of suspensions of isolated mito-
chondria were recorded at the wavelength range 515–700 nm (excited at 488 nm)
to monitor the presence and characteristics of PpIX after irradiation.
If not stated elsewhere, all experiments were performed at room temperature
(22 ± 2◦C).
Groups of data were checked for normal distribution by Shapiro–Wilk test. The
experiments were carried out in pair experiments and there for paired t-test was
used for parametrical analysis. Statistical significance for all tests was accepted at
the 0.05 level and lower.
Figure 1. Absorbance (at 560 nm) of
MTT assay products in non-irradiated
(control) and irradiated (laser) B-14
cells (A) and isolated rat heart mito-
chondria (B). Column heights repre-
sent mean ± S.D. (n = 18 for A and
n = 7 for B)
Kaššák et al.
To ensure that laser irradiation (532 nm, 30 mW, 1146 J/cm2) itself does not kill
the cells, survival of B-14 cells after laser irradiation was examined using Trypan
blue dye exclusion assay. There was no significant difference in the amount of dead
cells in non-irradiated and irradiated sample (data not shown).
Metabolic activity of B-14 cells after irradiation was estimated by MTT assay.
We found that laser illumination resulted in a significantly (p = 0.003) higher ab-
sorbance values (by 8%) of dissolved purple formazan crystals in irradiated samples
in comparison to non-irradiated controls (Fig. 1A).
Another step was to examine mitochondrial transmembrane potential of B-14
cells after exposure to green laser irradiation by means of the DiOC6(3) fluores-
cent probe. Laser irradiation of B-14 cells resulted in a statistically significant
(p = 0.017) increase (by 13%) in DiOC6(3) fluorescence intensity in comparison to
controls (Fig. 2).
MTT assay was also used in isolated mitochondria suspension to examine the
effect of green laser irradiation on stimulation of processes related to oxidative
phosphorylation. Production of formazan crystals from the MTT measured by ab-
sorbance at 560 nm was significantly (p = 0.033) higher (by 30.3%) in irradiated
mitochondria compared to controls (Fig. 1B).
Changes in mitochondrial membrane fluidity after irradiation were assessed in
terms of DPH fluorescence anisotropy. Kinetics of DPH incorporation was mon-
itored during 30 min following the addition of DPH to mitochondria suspension
Figure 2. Transmembrane potential of B-14 cells evaluated by fluorescence intensity
of DiOC6(3) (emission at 535 nm; excitation at 485 nm) in non-irradiated (control) and
irradiated (laser) samples. Values are shown as mean ± S.D. of 6 independent experiments.
Green Laser Effect on the Mitochondria
Figure 3. Time course of fluorescence anisotropy of DPH in the control, non-irradiated
mitochondria (dashed black line), and in mitochondria irradiated by green laser light
(solid grey line). The data presented are mean values of ten independent experiments;
S.E.M. did not exceed 6%. The minimum changes are not statistically significant.
Figure 4. Laser light-induced changes in intensity of the 635 nm fluorescence peak (ex-
citation at 488 nm) of isolated rat heart mitochondria. Data are shown as mean ± S.D.
of 5 individual measurements. Insert: fluorescence emission spectrum (excitation at 488
nm) of isolated rat heart mitochondria suspension in case of an irradiated (black line) and
non-irradiated (grey line) sample.
Kaššák et al.
for irradiated as well as non-irradiated samples (Fig. 3). As observed, laser ir-
radiation of isolated mitochondria suspension does not significantly change DPH
fluorescence anisotropy, indicating that mitochondrial membrane fluidity was not
affected by laser light.
Concurrent with fluidity measurements, fluorescence emission spectra of irra-
diated and non-irradiated mitochondria suspensions were recorded to establish the
fluorescence of PpIX and its possible changes. A representative example of fluores-
cence spectra of isolated rat heart mitochondria suspension, when illuminated or
kept in the dark, are presented in Fig. 4. Both spectra show fluorescence maximum
at 635 nm, corresponding to emission of PpIX. Laser irradiation of isolated mito-
chondria did not result in a shift of the peak position or spectral shape. The only
differences were in the values of fluorescence intensity, which showed a significant
decrease (by 20.7%, p = 0.022) in irradiated sample.
Laser induced changes in the biological tissues requires the absorption of the laser
light energy by a specific molecule. Only then the energy of laser irradiation may be
used as stimulus for reactions. The primary mechanism of light action after absorp-
tion of light quanta and the promotion of electrically excited states have not been
established. Possible explanations include stimulation of ascorbic acid uptake by
cells, stimulation of photoreceptors in the mitochondrial respiratory chain, changes
in cellular ATP or AMP levels, and cell membrane stabilization (Conlan et al.
In mitochondria, five possible mechanisms have been discussed in previous
studies (El Batanouny et al. 2002; Karu 2003) including singlet-oxygen hypothesis,
redox properties alteration hypothesis, nitric oxide hypothesis, superoxide anion
hypothesis and transient local heating hypothesis. However, these were postulated
for red and infra-red light operating lasers. Green laser light was not included, but
we may apply some of these hypotheses to explain or discuss our results.
In present work we showed that the irradiation by green laser significantly
increases (by 8%) the ability of the B-14 cells to reduce tetrazolium salt (MTT)
to formazan crystals (Fig. 1A). Reduction of MTT in isolated cells and tissues is
regarded as an indicator of cell redox activity. The reaction is attributed mainly
to mitochondrial enzymes and electron carriers (Bernas and Dobrucki 2002). Thus
we may suggest that the increase in the MTT degradation is closely related to the
positive stimulation of the mitochondrial enzymes and electron carriers by green
laser light. Suggestion is consistent with the results on mitochondrial transmem-
brane potential of B-14 cells monitored by the DiOC6(3) fluorescent probe as well
as on MTT assay carried out on isolated rat heart mitochondria.
We observed the significant increase in DiOC6(3) fluorescence intensity in ir-
radiated samples of B-14 cells (Fig. 2), which could be attributed to the increase
in mitochondrial transmembrane potential, as DiOC6(3) fluorescence intensity is
increased by the H+exchange between external glutamate and internal aspartate
Green Laser Effect on the Mitochondria
or cysteinsulfinate (Stipani et al. 1983). The mitochondrial transmembrane poten-
tial is a sensitive indicator for the energetic state of the mitochondria and therefore
the whole cells (Brand et al. 1994), and can be used to assess the activity of the
mitochondrial proton pumps, electrogenic transport system, and the activation of
the mitochondrial permeability transition (Zoratti and Szabo 1995). It is known
that mitochondria respiratory chain pumps protons outwards and therefore it cre-
ates positive transmembrane mitochondrial potential outside the inner membrane.
Although inhibition of oxidative phosphorylation in the presence of DiOC6(3)
has been reported (Rottenberg and Wu 1998), according to the literature, inhibitory
effect is not significant in the concentration used.
As we suggested above that observed effect of laser on whole cells is closely re-
lated to mitochondria, in next experiments we focus on investigation of laser effect
on isolated mitochondria. Irradiated isolated rat heart mitochondria degraded MTT
to formazan crystals by 30.3% in comparison to non-irradiated controls (Fig. 1B).
This increase is dramatic in comparison to slight 8% stimulation in B-14 cells and
was probably caused by the free exposure of the isolated material to the laser radi-
ation. The redox properties alteration hypothesis postulates that photoexcitation
of certain chromophores in the cytochrome c oxidase molecule (like CuAand CuB
or hemes a and a3) influences the redox state of these centres and, consequently,
the rate of electron flow in the molecule (Karu 2003). MTT degradation in the
mitochondria is performed by various enzymes in there, mainly by enzymes related
to oxidative phosphorylation.
The supply of energy by the mitochondrion depends on the maintenance of
the chemiosmotic gradient across its inner membrane (Mitchell 1979). This gra-
dient, also known as the proton motive force, is generated by three respiratory
enzyme complexes which use the free energy released during electron transport to
translocate protons from the mitochondrial matrix into the intermembrane space.
Proton motive force has two components: the mitochondrial membrane potential
(negative in matrix) and the pH gradient (alkaline in matrix) (Mathur et al. 2000).
Changes in mitochondrial transmembrane potential are integral to cell life. In nor-
mal cell function, the maintenance of transmembrane potential is essential for ATP
synthesis (Škárka and Ošťádal 2002).
As we presented, laser irradiation does not affect significantly the degree of
DPH fluorescent anisotropy (Fig. 3), which corresponds to unchanged ordering of
phospholipid molecules in the hydrocarbon region of membranes (lipid packing), i.e.
to unchanged mitochondrial membrane fluidity. However, we observed changes in
PpIX fluorescence intensity (Fig. 4). The prosthetic group of heme in cytochromes
b, c1and c is iron PpIX, the same heme as in myoglobin and hemoglobin (Berg et
al. 2002), which are reported to have high absorption of green light. PpIX is also
synthesized from 5-aminoleavulinic acid (5-ALA) in the mitochondria and then dif-
fuses into the cytoplasm of the cell (Peng et al. 1997; Tabata et al. 1997; Zhang et al.
2000). After absorption of laser light energy by PpIX, photobleaching could occur
due to its photodynamic action. However, the concentration of the PpIX in isolated
mitochondria is suggested to be very low, and thus the photodynamic effect has
Kaššák et al.
limited affection on membrane structure, and does not cause significant dysfunction
of the membrane and membrane bonded processes. Production of reactive oxygen
species has been reported (Oleinick et al. 2002) to cause the dissipation or collapse
of the mitochondrial transmembrane potential and decrease in the membrane flu-
idity (Ricchelli et al. 2001), what is not in agreement with our results. We have
found out insignificant changes of membrane fluidity but significant increase in the
mitochondrial transmembrane potential. These results does not support occurrence
of damaging photodynamic effect of PpIX in presence of laser irradiation.
All our experiments were performed at room temperature, which is below the
optimal temperature for living cells. We may only suppose that the observed effect
would be more notable in the proper temperature conditions.
In conclusion we can say that our experimental results proved that mitochon-
drial functional alterations occur in response to 532 nm low level laser irradiation.
Of these, in particular, the increase in mitochondrial oxidative phosphorylation pro-
cesses may indicate a biostimulating effect. However, further experiments exploring
applications of LLLT should follow.
Acknowledgements. We would like to thank to Prof. A. Ziegelhöffer and staff of Insti-
tute for Heart Research, Slovak Academy of Science (Bratislava, Slovakia) for the mito-
chondria isolation and material preparation. This study was supported by the University
of Lodz research grant No. 505/441, Slovak Grant Agency VEGA 1/0253/03 and APVT
51-013802. P. Kaššák was a recipient of the Jozef Mianowski Fellowship Fund (Warszaw,
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Final version accepted: February 11, 2005