Compact multi-functional skin spectrometry set-up

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1 Compact multi-functional skin spectrometry set-up Ilona Kuzmina *, Alexey Lihachev, Lasma Gailite and Janis Spigulis Bio-optics and Fibre Optics Laboratory, Institute of Atomic Physics and Spectroscopy University of Latvia, Raina Blvd. 19, Riga, LV-1586, Latvia ABSTRACT A portable fibre-optic spectrometry set-up has been assembled and tested for applications in skin diffuse reflectance spectrometry, laser fluorescence spectrometry and multi-wavelength reflection photoplethysmography (multi-ppg) studies. The spectrometry set was tested by diffuse reflectance and fluorescence measurements for diagnostics of skin vascular malformations and pigmented diseases such as nevi and melanoma. In addition, studies of microcirculation in blood vessels located at different depths from the skin surface were performed by the multi-ppg method. The results of skin diffuse reflectance and autofluorescence showed differences in spectra of healthy and pathologic skin. The parameter ratio pathologic/healthy has been calculated in order to check the possibility of quantifying the specific pathology. The autofluorescence fading effect was observed. Our studies of the blood volume pulsations confirmed the possibility to separate the time-variable PPG component from the total skin diffuse reflectance signal. Keywords: Spectroscopy, diffuse reflectance, autofluorescence, photoplethysmography 1. INTRODUCTION Human skin is characterized by absorption and strong light scattering properties. There are many kinds of chromophores in skin, but a few major chromophores predominantly determine the optical absorption within each skin layer. Melanin in epidermis and both oxy-haemoglobin and deoxy-haemoglobin in dermis are assumed as the primary skin chromophores [1, 2]. Oxy-haemoglobin absorbance has a strong maximum at 412nm (known as the Soret band) and maxima at 542 and 577nm (known as q-bands). Deoxy-haemoglobin absorbance exhibits a Soret band at 430nm and a single q-band at 555nm, and a low value in the red region, although higher than those of oxy-haemoglobin. Melanin absorption has no characteristic maximum in the visible region but demonstrates a monotonic increase toward shorter wavelength. 1,2 The scattering emerges from inhomogeneities in the refractive index that corresponds to microstructural inhomogeneities and it depends on the size and shape of the scatterers and the wavelength of the light. Typical scatterers in skin have dimensions in the range µm. 2 The constituents of skin that are strong scatterers include collagen and elastin fibres, erythrocytes, sub-cellular organelles (most notably pigmented melanosomes, nuclei, and mitochondria), and cell membranes. 1 Structures with dimensions greatly exceeding the light wavelength in epidermis (keratohyalin granules) and in dermis (collagen fibres) are responsible for highly forward directed scattering. Because epidermis is less thick than dermis, scattering in the epidermis is less important than dermal scattering when determining the penetration of optical radiation in skin. Dermal connective tissue is practically entirely responsible for the majority of light scattering in the skin. It also determines the diffuse pattern of light distribution within the skin and the formation of the backscattered diffuse reflectance. 2 Skin also contains various types of native fluorophores with unique absorption and emission spectra, different fluorescence quantum efficiency, fluorescence decay time and distribution within the skin. Some fluorophores have similar absorption and fluorescence spectra, and typically, fluorescence spectra measured on the skin surface are the result of the overlapping bands of various such fluorophores. The closer the excitation wavelength to the centre of the so-called therapeutic window ( nm) the larger is the penetration depth of the excitation light in tissue and the larger tissue volume is probed by the excitation light. As a result, new kinds of fluorophores located in deeper skin layers contribute to the total tissue fluorescence measured. 2 * kuzminailona@inbox.lv; phone/fax Advanced Optical Materials, Technologies, and Devices, edited by Steponas Asmontas, Jonas Gradauskas Proc. of SPIE Vol. 6596, 65960T, (2007) X/07/$18 doi: / Proc. of SPIE Vol T-1

2 This paper presents the portable spectrometry set-up for skin diagnostics. Our set-up is adapted for applications of four non-invasive spectroscopic methods: skin colour measurements, diffuse reflectance spectrometry, autofluorescence spectrometry and multi-wavelength reflection photoplethysmography. 2. THE SPECTROMETRY SET-UP The main parts of the spectrometry set are: light sources, spectrometer, skin contact probes, optical fibres and laptop computer (Fig.1). The set of light sources comprises halogen lamp (λ = nm) for diffuse reflectance measurements and several lasers - violet diode laser (405 nm), DPSS laser (532nm and 1064nm), red diode laser (645 nm), infrared diode laser (807 nm), GaAs infrared emitting diode (central wavelength 930 nm). The emission power delivered to the skin contact sites ranges between 1.6 mw to 17 mw for the various light sources listed above. SMA connector A Figure 1. Compact multi-functional skin spectrometry set. A-skin contact probe for diffuse reflectance and fluorescence measurements, B-skin contact probe for diffuse reflectance and multi PPG measurements. o. B Different optical fibre cables guided the light from source to skin and detector. Two types of skin contact probes were used. Fibre bundle of 7x400µm fibres (6 source fibres, 1 detector fibre, core diameter of each fibre - 400µm) combined with skin contact probe A was provided for diffuse reflectance and fluorescence measurements. The skin contact probe A was placed athwart to skin surface for diffuse reflectance measurements in the skin upper layer and at 45 0 angle for fluorescence and colour measurements. The dual skin contact probe B for diffuse reflectance measurements in skin deeper layers and multi-ppg measurements was used. In this case different fibre cables for delivery and collection were put together. Fibre with 600µm core diameter integrated with Y type fibre bundle of 3x200µm fibres was used for light delivery. Fibre cable of 7x200µm fibres (round to linear type) was used for signal detection. The distance between source and detector fibres was changed from 2 to 5mm in order to analyze different skin layers. As a detector the dual channel AvaSpec spectrometer with 2048 pixel CCD Detector Array, spectral range nm and resolution 2.1 nm was employed. The data storage and processing were performed by AvaSoft and originally developed software. AvaSoft programme provided data storage of colour, diffuse reflectance and fluorescence measurements. Original software was developed for multi-ppg experiments. This software ensures fast sequential readings of the whole spectra with the sampling rate 20 s -1. In result, a 3D-matrix (intensity-wavelength-time) has been created, and further intensity-time sections of the matrix have lead to extracted time-variable PPG signals at any fixed wavelength with temporal resolution 5 ms. 3 Proc. of SPIE Vol T-2

3 3. COLOUR MEASUREMENTS The International Commission on Illumination (Commission Internationale de l Eclairage CIE) established a tristimulus colour system based on a psycho-photometric method. 1 This tristimulus analysis converts intensity vs. wavelength data (i.e. spectral information) into three numbers that indicate how a colour of an object appears to a human observer. The CIE L* a* b* system has developed to be closely and linearly correlated with the response of the human eye. It expresses colour in the following parameters. L* indicates light intensity (quantity of reflected light weighted with the spectral response of the human eye) and takes values from 0 (black) to 100 (white). Parameter a* indicates the colour of the object on a scale that goes from green (negative values) to red (positive values). Criterion b* indicates the colour of the object on a scale from blue (negative values) to yellow (positive values). Axes a* and b* cross the L* axis at their zero values. This system has been used widely in the colour s study of different skin types and UV-induced tanning reaction b 6 Port-wine stain Skin PWS 4 2 -a a b Figure 2. Colour parameters of healthy skin and port-wine stain. 8 We took colour measurements on different kinds of vascular malformations. 8 Figure 2 presents colour parameters of port-wine stain and adjacent healthy skin. Port-wine stain shows higher a* coefficient values than healthy skin and value a=2 indicates the border between healthy skin and PWS. Parameter a* corresponds to redness of the colour and was mainly used for erythema evaluation by other authors DIFFUSE REFLECTANCE SPECTROSCOPY Diffuse reflectance method is based on measurements of the attenuation of the light intensity as it is remitted by the skin. As a light source a tungsten halogen lamp, deuterium lamp, xenon arc lamp or a broadband white LED usually are employed. This method uses fibres for light guidance and integration sphere or skin contact probe that come in contact with the skin surface. In the case of integration sphere, the delivery fibre illuminates some area of the surface and the detector fibre collects light from the entire area permitted by the aperture of the integrating sphere. The illuminated area is always smaller than the aperture of integrating sphere. Thus the total diffuse reflectance is measured. The skin contact probe is used for local diffuse reflectance measurements: light is delivered at some point on the skin surface and is collected at adjacent point or at some distance from source point. The fibre geometry is important as the fibre size and distance between illumination and collection fibres affect the sampling volume and the sampling depth. Skin contact probes are usually designed in such a way that minimizes pressure to the skin so that capillary constriction and skin blanching are avoided. Typically, prior to measurements the detector dark current is recorded, D, and a spectrum is acquired from the reflectance standard (defined as 100% at all wavelength), S ref (λ). 1 The diffuse reflectance spectrum of the skin site, R (λ), is then defined by the measured signal from the site, S (λ), corrected for the dark current and the reflectance standard: R (λ) = (S (λ) - D)/(S ref (λ) - D). Proc. of SPIE Vol T-3

4 Diffuse reflectance method was used to test skin pigmented lesions and vascular malformation by our spectroscopy set. Analysis of diffuse reflectance spectra helped to assess the pathologic skin by comparing its spectral data with those of the adjacent healthy skin. Before comparing each spectrum was normalized at particular wavelength. In the case of portwine stain normalization was made at 500nm in visible region and at 700nm in IR region. Spectra of nevi were normalized at wavelength with maximal measured intensity (approximately between nm). In addition the derivative and approximation of ratios (nevus norm / healthy norm ) were computing (results are not shown). 9 Thus the slope of curve was evaluated. Ratio(lesion norm /healthy norm ) 1,6 1,4 1,2 1,0 0,8 Port-wine stain Before treatment Immediatly after After several month Wavelength, nm Figure 3. Recovery pattern of one particular patient. 8 Figure 3 shows the relative changes in PWS diffuse reflectance spectra for one particular patient during treatment in visible spectral region. We observed that port-wine stains have oxy-haemoglobin absorption peaks in the wavelength range from 500nm to 600nm. Oxy-haemoglobin absorption strictly increased immediately after treatment by laser exposure. The spectrum of pathologic skin approached to spectrum of healthy skin after several months. Normalization at 500nm allows quantifying the content of oxy-haemoglobin. Port-wine stain Clark and dermal nevi Ratio(lesion norm /healthy norm ) 1,00 0,95 0,90 0,85 Before D=2mm After 0, Wavelength, nm D=0,4mm Ratio(lesion norm /healthy norm ) 3,0 2,5 2,0 1,5 1,0 0,5 Clarc hyperpigm Dermal hyperpigm Clarc mean pigm Dermal mean pigm Clarc less pigm Wavelength, nm Figure 4. Ratios of spectra before and after treatment in the IR range. 8 Figure 5. Ratios of spectra for different degree of nevi pigmentation. 9 Proc. of SPIE Vol T-4

5 We observed differences between healthy and pathologic skin in infrared region, too. Comparison of diffuse reflectance spectra at two distances between the source and detector fibres (D=0.4mm and D=2mm) before and after treatment is demonstrated in figure 4. 8 According to the data in the literature the longer source-detector distances correspond to deeper probed skin layers. Thus we assumed that distance between fibres D=2mm corresponds to 2mm depth in the skin and D=0,4mm - to superficial layers which depth is less than 2mm. 10,11 Diffuse reflectance spectra of PWS exhibit higher differences compared to healthy skin at deeper layers and recovery pattern one month after treatment at both sourcedetector distances. Measurements of pigmented skin lesions (nevi) manifest some correlation between slope of the spectral curve and pigmentation degree of nevi (Fig. 5.). 9 Higher pigmentation degree has greater slope parameter. Comparison of two kinds of nevi (Clark and dermal) shows higher slope parameter for Clark nevus that is considered dangerous, because it can transform to melanoma (skin cancer) with very high probability. Thus according to our research malignant skin lesions have higher slope parameter than benign malformations. However, the pigmentation factor is more prominent. 5. FLUORESCENCE SPECTROSCOPY AND MULTI-PPG The classic fibre optic probe to measure fluorescence consists of at least one excitation and one collection fibre. A shield placed at the distal end of the fibres enables the illuminated and probed area to overlap. The fraction of overlapping increases with enlargement of the numerical aperture of the fibres and the thickness of the shield. A typical shield thickness is 1 to 7 mm. If a shield is omitted, fluorescence can still be detected, but will originate from deeper layers because the average photon path length is increased. 13 Our equipment test results of autofluorescence showed differences in spectra of healthy and pathologic skin. 18,19 The autofluorescence fading effect was observed. Reflection photoplethysmography is a non-invasive method for studies of the blood volume pulsations by detection and analysis of the tissue back-scattered optical radiation. Blood pumping and transport dynamics can be monitored at different body locations - fingertip, earlobe, forehead, forearm, etc. with relatively simple and convenient PPG contact probes. 14 Usually the emitter in PPG probe is a narrow-band CW source like LED or laser, so the obtained biosignals are reflecting blood pulsations within a fixed penetration volume that is determined by the emitter wavelength. 15 Multispectral reflection PPG can give additional information on blood pulsations at different penetration depths correspondingly to the used wavelengths selection. 16,17 Studies of microcirculation in blood vessels located at different depths from the skin surface were performed by the multi-ppg method. Analysis of the blood volume pulsations confirmed the possibility to separate the time-variable PPG component from the total skin diffuse reflectance signal CONCLUSIONS Presented spectrometry set provides multifunctional skin spectroscopic investigation by colour, diffuse reflectance, fluorescence and multi-wavelength reflection photoplethysmography methods. It was tested by diffuse reflectance and fluorescence measurements for diagnostics of skin vascular malformations and pigmented diseases such as nevi and melanoma. The tests of blood volume pulsations were also made. These methods are promising in diagnostics of skin vascular and pigmented lesions and have potential for malignant tissue diagnostics. This work was mainly supported by European Social Fund. ACKNOWLEDGEMENTS REFERENCES 1. G. N. Stamatas, B.Z. Zmudzka, N. Kollias and J. Z. Beer, Non-invasive measurements of skin pigmentation in situ, Pigment Cell Res. 17, pp , V. V. Tuchin, Handbook of optical biomedical diagnostics, SPIE Press, Proc. of SPIE Vol T-5

6 3. J. Spigulis, L. Gailite, Multi-spectral Reflection Photoplethysmography: Potential for Skin Microcirculation Assessment, Tech. Digest, OSA 2006 Biomedical Optics conf., Fort Lauderdale, USA, J.K. Wagner, C. Jovel, H. L. Norton, E. J. Parra, M. D. Shriver, Comparing quantitative measures of erythema, pigmentation and skin response using reflectometry, Pigment Cell Res. 15, pp , G. Zonios, J. Bykowski, N. Kollias, Skin melanin, hemoglobin, and light scattering properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy, J. Invest. Dermatol. 117, pp , M. D. Shriver, E. J. Parra, Comparison of narrow-band reflectance spectroscopy and tristimulus colorimetry for measurements of skin and hair color in persons of different biological ancestry, Am. J. Phys. Anthropol. 112, pp , I. L. Weatherall, B. D. Coombs, Skin color measurements in terms of CIELAB color space values, J. Invest. Dermatol. 99, pp , I. Kuzmina, V. Gilis, A. Abelite, J. Spigulis, Diffuse Reflectance of Skin Vascular Malformations, Proc. ESBME S3 05, I. Kuzmina, L. Gailite, A. Lihachev, R. Karls, J. Spigulis, Diffuse Reflectance Spectroscopy of Skin Pathologies, IFMBE Proceedings 9, pp , I. Fridolin, L-G. Lindberg, Optical non-invasive technique for vessel imaging: I. Experimental results, Phys. Med. Biol. 45, pp , J. R. Mourant, T. M. Johnson, G. Los, I. J. Bigio, Non-invasive measurement of chemotherapy drug concentrations in tissue: preliminary demonstrations of in vivo measurements, Phys. Med. Biol. 44, pp , S. Takatani, Toward absolute reflectance oximetry: I. Theoretical consideration for non-invasive tissue reflectance oximetry, Adv. Exp. Med. Biol. 248, pp , U. Utzinger, R. R. Richards-Kortum, Fiber optic probes for biomedical optical spectroscopy, J. Biomed. Opt. 8, 1, pp , J. Spigulis, Optical non-invasive monitoring of skin blood pulsations, Appl. Opt. 44, pp , H. Ugnell, P. Å. Öberg, Time variable photoplethysmographyc signal: its dependence on light wavelength and sample volume, Proc. SPIE 2331, pp , M. Sandberg, T. Lundeberg, L. G. Lindberg, B. Gerdle, Effects of acupuncture on skin and muscle blood flow in healthy subjects, Eur. J. Appl. Physiol. 90, pp , N. D. Futran, B. C. Stack, Jr., C. Hollenbeak, J. E. Scharf, Green light photoplethysmography monitoring of free flaps, Arch. Otolaryngol. Head Neck Surg. 126, pp , A. Lihachev, J. Spigulis, Skin autofluorescence fading at 405/532 nm laser excitation, Tech. Digest, Northern Optics 2006, Bergen, Norway, p. 143, A. Lihachev, J. Spigulis, Human skin fluorescence: intensity fading effects at 405 nm and 532 nm laser excitation, Latv. J. Phys. Techn. Sci. 2, p , Proc. of SPIE Vol T-6

Laser- and Light-Induced Autofluorescence Spectroscopy of Human Skin in Dependence on Excitation Wavelengths

Laser- and Light-Induced Autofluorescence Spectroscopy of Human Skin in Dependence on Excitation Wavelengths Vol. 112 (2007) ACTA PHYSICA POLONICA A No. 5 Proceedings of the International School and Conference on Optics and Optical Materials, ISCOM07, Belgrade, Serbia, September 3 7, 2007 Laser- and Light-Induced