Skin autofluorescence photo-bleaching and photo-memory
Alexey Lihacheva, Janis Lesinsa, Romualdas Rudysb, Saulius Bagdonasb and Janis Spigulisa
a
Institute of Atomic Physics and Spectroscopy, Univiversity of Latvia, Raina Blvd. 19, Riga,
Latvia, LV-1586;
b
Vilnius University, Laser Research Center, Sauletekio ave, 9, c. 3, Vilnius, Lithuania, LT-10222
ABSTRACT
Photo-bleaching of in-vivo skin autofluorescence intensity under continuous low power laser irradiation has been
studied. Fiber optic spectrometry set was used at continuous 405 nm and 532 nm laser irradiation. The fluorescence
bleaching curves during skin irradiation have been obtained and analyzed. Skin photo-memory effect showing signs
of low-power laser irradiation on in-vivo skin is discussed, as well.
Keywords: in vivo skin, photobleaching, autofluorescence.
1. INTRODUCTION
Photo-bleaching is a process of fluorescence intensity decrease during a lasting optical excitation. The skin
autofluorescence photo-bleaching (AFPB) was examined several times in the recent years. Most of the authors stated
that fluorescence intensity decreases in two phases and can be well described by double exponential equation. Some
remaining intensity usually exists, tending to a constant level after some time. AFPB has been observed at various
laser excitation wavelengths - ultra-violet (337 nm), violet (405 nm), blue (442 nm), green (532 nm) and red (632
nm), both under continuous and pulsed excitations in a wide range of power densities (1 ... 500 mW/cm2) [1-3].
Skin fluorescence photobleaching may find clinical applications in photodynamic therapy, skin keratinoid
concentration determination, sensitizing agent controlling, etc. [4-8]. Mechanism of the skin AFPB effect has not
been explained in details so far, but several hypotheses are examined experimentally. Low power laser irradiation
may cause direct skin fluorochrome (for instance, porphyrin) degradation, but also some multi-step photoprocesses
cannot be excluded.
One should note that AFPB of healthy skin takes place at very low laser power densities, even lower than those fixed
for skin at the European laser safety standard (200 mW/cm2, exposition time to 103 s., minimal allowed dose 20.6 J)
[8].
This study continues our previous research [9-11] with the aim to understand more deeply the tissue AF bleaching
effects under low-power continuous laser irradiation at two visible wavelengths – 405 nm and 532 nm.
2. METHODS AND EQUIPMENT
The measurement setup comprises a cw laser (405 nm or 532 nm) with focusing output SMA-connector, Y-shaped
optical fiber bundle for delivery of the laser radiation to the skin probe and the fluorescent light to spectrometer (via a
laser-blocking filter) which was connected to PC. The emitting and detecting fibers were placed 3 mm from the skin
surface and sloped at 45 degree angle. The typical excitation time was 2-3 minutes, the spectrometer integration time
0.5 sec, laser power density on the skin ~85mW/cm2, area of the excited skin surface ~ 0.3cm2 [9]. During the skin
recovery measurements, 532 nm laser excitation for periods of 1 minute was used to bleach the AF on forearm, with 1
minute intervals between excitations. Overall 3 volunteers of skin phototype III and IV participated in the trial. In the
studies of laser signatures on skin, additionally 405 nm laser irradiation at power density ~35 mW/cm2 was used.
3. RESULTS AND DISCUSSION
Fig.1 presents healthy skin AF intensity decay detected simultaneously at two wavelengths under continuous 532 nm
laser excitation. More rapid bleaching at 600 nm was observed. The AFPB effect of healthy skin if the same place is
repeatedly excited by 532 nm laser is illustrated at Fig. 2. The fluorescence intensity exponentially decreases during
irradiation periods; however, after the „rest” between two excitations the AF intensity does not return back to the
initial level, but it is just slightly higher than at the moment of switching off the previous radiation. This indicates to
photo-induced irreversible processes in skin, or skin “photo-memory”. Our results show that it is a long-term effect –
the intensity of autofluorescence recovers to ~ 80% of its initial level only after three to five days (Fig. 3).
1,05
Fluorescence intensity, rel. u.
1,00
1- 680 nm
2- 600 nm
0,95
0,90
0,85
0,80
0,75
0,70
1
0,65
2
0,60
0,55
0,50
0
120
240
360
480
600
Time, s
Fluorescence intensity, rel.u.
Fig.1 Fluorescence intensity bleaching under continuous 532 nm laser excitation (outer skin, power density ~85 mW/cm2).
1- 680 nm
2- 600 nm
1,0
0,8
1
2
0,6
0
120
240
360
480
600
Time, s
Fig.2 The changes of AF intensity under repeated 532 laser excitations of the outer skin area. The spectra are normalized to
the initial fluorescence intensity.
100
Recovery of AF intensity (%)
80
60
40
20
0
0
25
50
75
100
125
Time (Hours)
Fig. 3 AF recovery dynamics of autofluorescence intensity after two minutes of 532 nm laser exposure on forearm at power
density 85 mW/cm2. The points with error bars represent the mean values for all volunteers.
Figure 4 illustrates two signatures of low-power laser radiation on human skin obtained in experiments with cruciform
masks and well-filtered fluorescence detection. In the case of previous irradiation of skin with cruciform shield (Fig.4,
a), the “fresh” skin exhibited more intense fluorescence after removal of the mask, while the pre-radiated opening of
similar shape emitted weaker after removal of the shielding mask (Fig.4, b).
a
b
1 cm
Fig. 4. Filtered AF images of normal skin under 405 nm laser irradiation and 35 mW/cm 2 power density. a) – fluorescence
image with permeable cross mask, b – fluorescence image of the same skin area without mask which was obtained 24 hours after
irradiation.
4. CONCLUSIONS
Results of the present study show unexpected sensitivity of in-vivo human skin to low power visible laser irradiation
at power densities 30-85 mW/cm2, i.e. well below the European skin safety limits. Autofluorescence bleaching is
responsible for skin “photo-memory” effect at these power levels. Since the mechanism of skin AFPB is not yet fully
clear, in future more efforts should be put to study this phenomenon more deeply. It might open new possibilities for
skin assessment and eventually lead to revision of laser skin safety standards.
ACKNOWLEDGMENTS
The financial support of European Social Fund (grant #2009/0211/1DP/1.1.1.2.0/09/APIA/VIAA/077) is highly
appreciated.
REFERENCES
1. H. Zeng, C. E. MacAulay, B. Palcic, and D. I. McLean. Laser induced changes in autofluorescence of in-vivo
skin, Proc. SPIE, Vol. 1882, 278–290 (1993).
2. A. Stratonnikov, V. S. Polikarpov, and V. B. Loschenov. Photobleaching of endogenous fluorochroms in tissues
in vivo during laser irradiation, Proc. SPIE, Vol. 4241, 13–24 (2001).
3. E. V. Salomatina and A.B. Pravdin. Fluorescence dynamics of human epidermis (ex vivo) and skin (in vivo), Proc.
SPIE, Vol. 5068, 405–410 (2003).
4. A. V. Ryabova, A. A. Stratonnikov, V. B. Loshchenov, Laser spectroscopy technique for estimating the efficiency
of photosensitisers in biological media, Quantum Electron, 36 (6), 562–568 (2006).
5. Finlay, J.C., et al., Photobleaching kinetics of Photofrin in vivo and in multicell tumour spheroids indicate two
simultaneous bleaching mechanisms. Phys Med Biol, 49(21), 4837-60 (2004).
6. M.E. Darvin, N.N. Brandt, J. Lademann. Photobleaching as a method of increasing the accuracy in measuring
carotenoid concentration in human skin by Raman spectroscopy. Opt. Spectrosc. 109(2): 205-210, (2010).
7. V. Tuchin. Handbook of Optical Biomedical Diagnostic, SPIE-The international Society for Optical Enginering
P.O. Box10, 709-877 (2002).
8. Safety of laser products –Part 1: Equipment classification and requirements, IEC 60825-1 (2007).
9. A. Lihachev, J. Spigulis. Skin autofluorescence fading at 405/532 nm laser excitation, IEEE Xplore, 10.1109/NO,
63-65 (2007).
10. A. Lihachev, J. Lesins, D. Jakovels, J. Spigulis. Low power cw-laser signatures on human skin. Quantum
Electron, 40 (12), 1077–1080 (2010).
11. J. Spigulis, A. Lihachev, and R. Erts. Imaging of laser-excited tissue autofluorescence bleaching rates, Appl
Optics, Vol. 48, Issue 10, D163-D168 (2009).