Skin Research and Technology 2014; 0: 1–10 Printed in Singapore  All rights reserved doi: 10.1111/srt.12129

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Skin Research and Technology

Optical coherence tomography using images of hair structure and dyes penetrating into the hair Tetsuya Tsugita1,2 and Toshiaki Iwai2 1

Skin Beauty Research Laboratories, Kao Corporation, Tokyo, Japan and 2Division of Bio-Applications and System Engineering (BASE), Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan

Background/purpose: Hair dyes are commonly evaluated by the appearance of the hair after dyeing. However, this approach cannot simultaneously assess how deep the dye has penetrated into hair. Methods: For simultaneous assessment of the appearance and the interior of hair, we developed a visible-range red, green, and blue (RGB) (three primary colors)-optical coherence tomography (OCT) using an RGB LED light source. We then evaluated a phantom model based on the assumption that the sample’s absorbability in the vertical direction affects the tomographic imaging. Results: Consistent with theory, our device showed higher resolution than conventional OCT with far-red light. In the experiment on the phantom model, we confirmed that the tomographic imaging is affected by absorbability unique to the

sample. Furthermore, we verified that permeability can be estimated from this tomographic image. We also identified for the first time the relationship between penetration of the dye into hair and characteristics of wavelength by tomographic imaging of dyed hair. Conclusion: We successfully simultaneously assessed the appearance of dyed hair and inward penetration of the dye without preparing hair sections.

tomography (OCT) is a technique used for imaging the tomographic structure of living tissue, first reported by Huang et al. (1). Following imaging of the eye retina and iris (2, 3), the scope of imaging has been extended to the skin (4, 5). In 2001, SKINDex 300 (ISIS Optronics GmbH, Mannheim, Germany) was released and used as an OCT device exclusively for imaging of the skin. This system uses eight LED light sources, each with a center wavelength of k0 = 1300 nm, and is based on the time-domain method. Its optical axial resolution [FWHMint] is 7.4 lm, and the numerical aperture of the focusing lens is NA = 0.19. Thus, the diffraction-limited lateral resolution [FWHMFoc] is 4.5 lm. This system produces a two-dimensional image (lateral direction of 1 mm and an axial range of 0.9 mm) in 2 s (6), making it possible to perform imaging from the surface of the skin to the epidermis and the upper dermis without biopsy (7). The system can thus measure the thickness of the epidermis (8), which could be observed only by biopsy previously, as well as

structural changes in the same site (9, 10), which could not be determined by biopsy. Furthermore, the system indicates not only the structure of the skin but also the refractive index in the depth direction (11, 12), which is one of the optical characteristics, and has yielded novel findings. Subsequent studies have reported on tomographic imaging of dermal appendages, imaging of fingertip sweat glands (13), from nail plate to cuticle (14, 15), and the density and occupancy of the hair infundibular part (16). Many of these reports on appendages focused on verifying the accuracy of the system and included relatively little information. Hair, especially that of the head, is a dermal appendage that differs by person and race. It has a mean diameter of about 80–100 lm. It has a triplex structure. The medulla located in the center of the hair has a mean diameter of about 10 lm, or there may be no medulla. A cortical substance called cortex circumvolutes the medulla and is surrounded by cuticle on the outermost side, consisting of about three layers

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PTICAL COHERENCE

Key words: OCT – RGB light sources – LED – hair dyes – hair structure image

Ó 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Accepted for publication 4 January 2014

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Tsugita and Iwai

Fig. 1. Structure and parts of human head hair.

each with a thickness of about 0.5–1.0 lm (Fig. 1). In recent years, coloring dyes for dyeing head hair in a variety of colors for fashion or for changing age-related white hair into black hair to give a younger appearance have been put on the market. These formulations are evaluated based on the degree of penetration into the hair and the appearance of the finished color. The only presently available method for evaluating the penetration and appearance of finished color is to prepare sections of hair. However, the preparation of sections involves many problems; for example, the solvents used for preparation may affect the dyes, cause deformation of the hair itself, and take much time. In addition, the mechanism by which the appearance of finished color changes, as hair is repeatedly washed and as time advances, as well as how the dyes that have penetrated into the hair change, is not well understood, as the same site cannot be observed by preparing sections. Accordingly, there is no report on the structure of hair, penetration of coloring dyes, and estimation of color, using OCT. It is expected that by shortening the wavelength of the light source used for OCT and selecting the three primary colors of red, green, and blue (RGB) may not only increase the resolution in the optical axial direction but also clarify the relationship between the appearance of finished color and changes in dyes. The primary objective of this study was to develop a time-domain OCT system using visible LED light sources to achieve a higher resolution by shortening the central wavelength. The second objective was to examine the use of phantom models to clarify the effect of the absorption characteristics of a specimen on imaging using the three RGB wavelengths. The third objective was to estimate the transmittance of a specimen based on OCT imaging.

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Fig. 2. Experimental setup (full-field OCT).

In this study, a high-resolution OCT system using three RGB primary color LED light sources was developed; tomographic imaging of dyed hair was performed; and the relationship between images of dyes penetrating into the hair and the wavelength characteristics of the dyes was examined.

Material and methods Experiment system The optical system of the constructed microscope-type time-domain (TD-OCT) is shown in Fig. 2. The interference optical system is based on a Michelson interferometer. A specimen (S) is placed on one side. Light from the low coherent light source (L) is guided by fibers and separated by a beam splitter into the directions of the specimen and of the reference mirror. Each light beam is changed into parallel light by lens 1 (L1) and lens 2 (L2), and is irradiated to the specimen and the reference mirror (M). The reflected light from each surface is recombined by the beam splitter and measured with the detector (D). The interference signal can be obtained only when the difference in light path (iL) DL between the light path on the specimen side (ls) and the light path on the reference mirror side (lr) is zero: DL ¼ 2jlr  ls j

ð1Þ

where DL is the difference in light path between the light path on the specimen side (ls) and the light path on the reference mirror side (lr). We obtained this light path difference by scanning the movable sample stage. Moreover, the optical axial resolution (lc) is obtained by:

RGB-OCT imaging of colored hair

lc ¼

2ln2 k20 p Dk

ð2Þ

where lc, Dk, and k0 are the optical axial resolution (coherence length of the light source), wavelength bandwidth, and central wavelength, respectively. The optical axial resolution (lc) is in inverse proportion to Dk and is proportional to the square of the central wavelength k0. Lateral resolution Dx is determined by the numeric aperture of the lens and the bandwidth of the light source, and is given by Dx ¼

0:61  k0 NA

ð3Þ

where k0 is the source central wavelength and NA is the numeric aperture. It is inversely proportional to the numeric aperture of the lens NA and proportional to central wavelength k0. Wavelength property of phantom models When light is irradiated to a scattering medium with the thickness of Z, the light is affected by the medium and attenuates. The relationship between the intensity of incident light Iz and the intensity of transmitted light I0 is, according to Lambert–Beer’s law, given by: Iz ¼ I0 expðlt ZÞ

ð4Þ

where lt is the attenuation coefficient, which is the sum of the scattering coefficient ls and the absorption coefficient la, and is given by: lt ¼ ls þ la

ð5Þ

In this study, to change the absorption coefficient la, colored cellophane films were used for making phantom models. Estimation of transmittance from the OCT interference signal The distance from the surface (reference plane) to the glass surface is set as the reference distance (L), and for the phantom models, colored cellophane films are inserted between them. In the phantom models, each of the distance from the reference plane to the surface of the cellophane (L1), the thickness of the cellophane (L2), and the distance from the backside of the cellophane to the glass (L3) match the reference

distance (L). In OCT tomographic imaging, the thickness of the specimen is obtained as the optical distance (nL2), and the position of the glass surface (d) will shift and become as follows: L2 ¼ nL2  d

ð6Þ

where (d) and (nL2) can be read out from topological images. From Eq. (4), in the phantom model placed on glass, which was measured this time, it is considered that the light that transmits through the phantom once and is reflected by the glass surface with respect to the incident light (I0) is affected by the absorption characteristics of the phantom model again, giving: I ¼ I0 T 2

ð7Þ

Equation (7) reduces to sffiffiffiffi I T¼ I0

ð8Þ

where I, I0 and T are transmitted light intensity, incident light intensity, and transmittance, respectively.

Resolution of experiment system As the interference signal detector, a visiblelight highly sensitive CCD camera with peak sensitivity at 450 nm was used. Scanning in the depth direction was performed, with a fixed reference mirror, by shifting the specimen stage (Sigma Co., Tokyo, Japan) in the optical axial direction. Lenses 1 and 2 were objective lenses with NA of 0.50 (Olympus Corporation, Tokyo, Japan), as shown in Fig. 2. The three LED light sources used this time had a central wavelength of the three primary colors of 444.6 nm and a bandwidth [FWHM] of 21.8 nm (blue); a central wavelength of 522.6 nm and a bandwidth [FWHM] of 40.1 nm (green); and a central wavelength of 637.3 nm and a bandwidth [FWHM] of 18.4 nm (red). The reference SLD light source had a central wavelength of 846.0 nm and a bandwidth [FWHM] of 26.0 nm (Fig. 3). Interference signals obtained with this system are shown in Fig. 4. The optical axial resolution [FWHM] was calculated to be 3.25 lm (blue),

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Tsugita and Iwai

Fig. 3. Central wavelengths of LED and SLD.

2.70 lm (green), 8.22 lm (red), and 9.30 lm (SLD) from these interference signals when a Gaussian distribution was assumed. The optical axial resolution of the green LED light source having a central wavelength of 522.6 nm was calculated to be higher than that of the blue LED light source having a central wavelength of 444.6 nm, and the resolution (2.70 lm) was the highest among the three colors. This was due to the fact that the green LED light source had a wavelength width of 40.1 nm, which was twice as large as that of the blue LED light

source, thus the wavelength bandwidth Dk affects the resolution. Moreover, the respective resolutions in the horizontal direction were 0.54, 0.64, 0.78, and 1.03 lm. The resolution in the side direction has a wavelength dependency called diffraction limit, and in this system, the resolution in the side direction was 1.6 lm (actual measured value) determined from the CCD pixel size (14 9 14 lm) of the detector (manufactured by Bitran Corporation, Saitama, Japan) shown in Table 1. Shortening the central wavelength improves the theoretical resolution. When the central wavelength was 444.6 nm, the resolution was 3.25 lm. Moreover, even when the wavelength width is long, the theoretical resolution will be improved. The green LED light source having a central wavelength of 522.6 nm and a wavelength width of 40.1 nm, which was twice as large as that of the blue LED light source, had the highest resolution of 2.70 lm among the three colors.

Results and discussion Measurement of transmittance of the phantom model With respect to the three visible-light LED light sources having different central wavelengths,

(a)

(b)

(c)

(d)

Fig. 4. (a) Interfering signal of blue LED light source. S indicates side-lobe. (b) Interfering signal of green LED light source. (c) Interfering signal of red LED light source. (d) Interfering signal of SLD light source.

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RGB-OCT imaging of colored hair TABLE 1. Specifications of LED light source of each wavelength

Central wavelength (nm)

LED (blue) 444.6

LED (green) 522.6

LED (red) 637.3

SLD 846.0

Bandwidth (FWHM) (nm) Axial resolution (FWHM) (lm) Transverse (lm)

21.8 3.3 0.5

40.1 2.7 0.6

18.4 8.2 0.8

26.0 9.3 1.0

Fig. 5. Measurement of transmittance of samples by spectrophotometer.

Fig. 7. Each cellophane film was placed on the same glass plate. The OCT image shows each surface and the back side corresponding to the absorption characteristic of the sample, for each LED light source. 1) The surface of each cellophane; 2) the back side of blue cellophane; 3) the surface of glass.

Fig. 6. Transmittance characteristics in visible-light range of each cellophane.

we measured the transmittance of the blue, green, and red cellophane films with thickness of 17.5 lm to determine the effect of the absorption characteristics of a specimen in the optical axial direction (Fig. 5). The transmittance of each cellophane film was obtained for each wavelength using a spectroscopic detector (Ocean Optics, Inc., Dunedin, FL, USA) by irradiating light emitted from a white light source (Figs 6 and 7). The blue cellophane showed a transmittance of 0.65 at 425 nm when the measurement was started, and showed the highest transmittance of 0.68 at 470 nm. It attenuated steadily thereafter and maintained a low value

of 0.23 in the range from 603 to 658 nm and then increased again. The green cellophane transmitted most of the light (transmittance of 0.07) at 425 nm, the wavelength at which the measurement started, and showed a maximum value of 0.47 at 520 nm, showing the same trend as the blue cellophane with a transmittance of about 0.1 lower than that of the blue cellophane. The red cellophane perfectly absorbed light up to 580 nm and then transmitted light steadily, with the transmittance maintaining a value of 0.85 after 640 nm. The wavelength characteristics of each film closely matched the appearance.

OCT Imaging obtained from the absorption characteristics of each of the LED light sources and the phantom models Subsequently, the measured blue, green, and red cellophane films were placed on the same glass plate, and phantom models having different absorption coefficients la were constructed. These phantom models were then measured

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Tsugita and Iwai (a)

(b)

Fig. 8. (a) The optics model of the measured phantom models. (b) The optics model of the OCT tomographic images. According to Lambert–Beer’s Law, I light is transmitted twice the distance (there and back) inside the cellophane film. This shows the value of the incident light, which transmits through the cellophane film once, is reflected on the mirror surface, and is absorbed by the cellophane film again, then is detected. Where I0, I, and T are incident radiation intensity, transmitted intensity, and transparency, respectively.

using three kinds of LED light source in the optical axial direction. The imaging results are shown in Fig. 8. When using the blue LED light source having a central wavelength of 444.6 nm, interference signals were obtained from the front and rear surfaces of the blue cellophane only and additionally from the glass surface. As for the other green and red cellophane films, interference signals were obtained only from their front surfaces. This is considered to be because the blue cellophane transmits light and the green and red cellophanes absorb the wavelength of the light sources due to the central wavelength and transmittance of the phantom models, as shown in Fig. 7. Next, using the green LED light source having a central wavelength of 522.6 nm, interference signals were obtained from the front and rear surfaces of the blue and green cellophanes, but only from the front surface of the red cellophane. Moreover, in case of using the red LED light source having a central wavelength of 637.3 nm, interference signals were obtained from the front and rear surfaces of the red cellophane only. In other words, the OCT imaging in the optical axial direction attains full detection as strong interference signals are obtained from the surface of the object to be measured due to the difference of refractive index of the air layer and the surface of the object. However, it was demonstrated that deeper imaging in the optical axial direction was affected by the inherent wavelength characteristics of the specimen, especially absorption, and interference signals from the rear surface may not be obtained depending on the level thereof. As a result, it

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was suggested that for imaging of the inside of a specimen, it is necessary to take into account the inherent wavelength characteristics of the specimen and to select a suitable light source for the specimen.

Measurement results through OCT and estimation of transmittance of respective phantoms The thickness of the specimen is obtained as an optical distance by OCT. It was presumed that Lambert–Beer’s law is valid, and the transmittance of respective phantom models was obtained from the interference signal intensity in the axial direction obtained from OCT (Fig. 9). The results obtained by calculating from the OCT image (signal intensity) for the three kinds of LED light source are shown in Fig. 10 with

Fig. 9. The three lines are the actual measured values of each cellophane. The dashed lines are calculated from the signal intensity of OCT.

RGB-OCT imaging of colored hair (i)

(iii) a

(ii)

(i)

(ii)

b (iii)

(a) c

(b) d

(a)

(c)

(b)

(d)

Fig. 10. (a) Optical microscope images measured in the same site. Cu, cuticle; Co, cortex; Me, medulla. (b) Image of measurement. Goat hair dyed with red dye. (c) Tomographic images of goat hair dyed with red dye through longitudinal-direction OCT. (i) 444.6 nm, (ii) 522.6 nm, (iii) 637.3 nm, and (iv) 846.0 nm. (c)

broken lines. The error bar indicates standard deviation. As a result, as the actual measured values showed the same tendency, it was confirmed that the transmittance can be estimated from the tomographic images, and hence the color can be identified from the signal intensity of OCT. For practical use, transmittance must be measured taking into account the shape of the specimen and solvents. Here, we examined whether the transmittance of a specimen could be measured from the OCT signal intensity, and found that it is possible without the conventional complicated apparatus. This reduces the time and effort required for processing the specimen, avoids changes to the specimen caused by the solvents, and shortens the measuring time, which are very useful benefits. In addition, it was suggested that imaging much closer to observation by the naked eye is possible by utilizing wavelength characteristics in the visiblelight region. We verified the wavelength dependency of the central wavelength and the specimen using the visible LED light source and the phantom models. We consider that these results will be useful to industry as this method can quantitate, for

(d)

Fig. 11. (a) Optical microscope images measured in the same site. Cu, cuticle; Co, cortex; Me, medulla. (b) Goat hair dyed with a blue dye. (c) OCT tomographic image with the central wavelength of (i) 444.6 nm, (ii) 522.6 nm, (iii) 637.3 nm, and (iv) 846.0 nm.

example, from where a dyed object using a dye started to be dyed and from where it fades as a distribution in the optical axial direction.

Hair structure and dyes penetrating into hair imaging by OCT The characteristics of spectral reflectivity of blue dyes and red dyes were measured. Goat hair dyed with these two color dyes was prepared. Goat hair was selected as it is known to have a similar structure to human hair, taking into account usage for biological samples. The goat hair was placed on white paper and measured with OCT. For comparison with the real substance, sections were prepared by embedding with Sunp plates. The hair was sliced with the thickness of 4.0 lm in the longitudinal direction, and in the vertical direction (cross-sectional direction) with the thickness of 15.0 lm to enable observation of the penetration of dyes. Images of

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Tsugita and Iwai (i)

(ii)

(a)

(iii)

(b) (a)

(c)

(b)

(d)

Fig. 12. (a) Optical microscope images measured in the same site. Cu, cuticle; Co, cortex; Me, medulla. (b) Goat hair dyed with red dye. (c) OCT tomographic image with the central wavelength of (i) 444.6 nm, (ii) 522.6 nm, (iii) 637.3 nm, and (iv) 846.0 nm.

these sections and OCT images were compared. By comparing images of red-dyed hair in the longitudinal direction, the structure of the hair was verified, and by comparing images of redand blue-dyed hair in the cross-sectional direction, penetration of dyes and wavelength characteristics of dyes were examined. Tomographic images of goat hair dyed with a red dye through longitudinal-direction OCT for each wavelength light source are shown in Fig. 10. The three kinds of visible LED light source clearly show the boundary between the surface of the cuticle and cortex, the boundary of the cortex on the cardboard side, and the position of the backside cuticle, respectively. The medulla in the center of the hair was confirmed at each wavelength in the same way as for optical microscope images. Sites with and without medulla were revealed in OCT images at each wavelength, and agreed with those in the optical microscope images. It was confirmed

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Fig. 13. Reflectance (solid line) of the dye measured with spectral photometer. (a) Goat hair dyed with blue dye. (b) Goat hair dyed with red dye.

by observation that in the longitudinal direction, regarding the structure of goat hair, optical microscope images and OCT images were similar. However, near-infrared light had lower resolution in the optical axial direction than other light and showed larger thickness of surface cuticle than the measured one and an unclear boundary between the cardboard in the backside. In addition, the S on the surface of blue is a side-lobe, and its appearance and inhibition methods have already been reported (17–19). Figures 11 and 12, respectively, show a crosssectional image with the optical microscope and a tomographic image with the OCT, indicating the appearance of the finished color of goat hair when dyed with blue and red dyes. In the case of the blue and red dyes, tomographic images of the three kinds of visible LED light source showed that the dye

RGB-OCT imaging of colored hair

penetrated from the cortex slightly inside the surface cuticle, and this result was confirmed to be similar to that of optical microscope images. As to why it was not possible to image the side of the hair, it is considered that spatial coherence was lowered due to the angle of the specimen. Spectroscopic-measured reflectance of the hair and reflectance calculated from OCT images are shown with bands in Fig. 13. Reflectance calculated from tomographic images and spectroscopic-measured reflectance (shown by solid line) had the same tendency, and it was confirmed that colors can be estimated from tomographic images also in a biological sample. From the width (D: depth reached) of the interference signal from the cardboard at each wavelength including SLD, it is obvious that as the wavelength of light becomes larger, the depth reached by the dyes increases. Therefore, it is natural that red light enables imaging of a larger area than blue light.

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Conclusion In this study, a TD-OCT system using three RGB primary color LED light sources was conducted. High resolution was achieved by shortening the central wavelength. It was found that absorption characteristics and imaging were correlated, which is considered to be due to the transmittance of the specimen based on OCT imaging. From tomographic imaging of dyed hair, the relationship between dye penetration into hair and the wavelength characteristics of dyes was revealed. As a result, the reflected light characteristics of OCT images at each wavelength agreed with those of dyes in the visible-light region, and we succeeded in comparing and verifying a biological sample under an optical microscope. It is suggested that this method may help to clarify the variation per day of dyes.

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13. Saigusa H, Ueda Y, Yamada A, Ohmi M, Ohnishi M, Kuwabara M, Harun M. Maximum-intensityprojection imaging for dynamic analysis of mental sweating by optical coherence tomography. Appl Phys Express 2008; 1: 098001–098003. 14. Watanabe Y, Yamada K, Sat M. In vivo non-mechanical scanning grating-generated optical coherence tomography using an InGaAs digital camera. Opt Commun 2006; 261: 376–380. 15. Mogensen M, Thomsen JB, Skovgaard LT, Jemec GBE. Nail thickness measurements using optical coherence tomography and 20MHz ultrasonography. Br J Dermatol 2007; 157: 894–900. 16. Hori Y, Yasuno Y, Sakai S et al. Automatic characterization and segmentation of human skin using three-dimensional optical coherence tomograph. Opt Express 2006; 14: 1862–1877. 17. Zhang Y, Sato M, Tanno N. Resolution improvement in optical coherence tomography by optimal synthesis of light-emitting diodes. Opt Lett 2001; 26: 205–207. 18. Zhang Y, Sato M, Tanno N. Numerical investigations of optimal synthesis of several low coherence sources for resolution improvement. Opt Commun 2001; 192: 183–192.

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Tsugita and Iwai 19. Tripathi R, Nassif N, Nelson JS, Park BH, Boer JF. Spectral shaping for non-Gaussian source spectra in optical coherence tomography. Opt Lett 2002; 27: 406–408.

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Address: Tetsuya Tsugita Skin Beauty Research Laboratories, Kao Corporation, 2-1-3 Bunka, Sumida-ku, Tokyo 131-8501 Japan

Tel: +1 81-3-5630-9480 Fax: +1 81-3-5630-9341 e-mail: [email protected]

Optical coherence tomography using images of hair structure and dyes penetrating into the hair.

Hair dyes are commonly evaluated by the appearance of the hair after dyeing. However, this approach cannot simultaneously assess how deep the dye has ...
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