Full-depth epidermis tomography using a

Mirau-based full-field optical coherence

tomography
Chien-Chung Tsai,1 Chia-Kai Chang,1 Kuang-Yu Hsu,1 Tuan-Shu Ho,1 Ming-Yi Lin,2
Jeng-Wei Tjiu,2,4 and Sheng-Lung Huang1,3,*
1
Graduate Institute of Photonics and Optoelectronics, National Taiwan University, Taipei 10617, Taiwan
2
Department of Dermatology, National Taiwan University Hospital and College of Medicine, National Taiwan
University, Taipei, Taiwan
3
Department of Electrical Engineering, National Taiwan University, Taipei 10617, Taiwan
4
jengweitjiu@gmail.com
*
shuang@ntu.edu.tw

Abstract: With a Gaussian-like broadband light source from high

brightness Ce3+:YAG single-clad crystal fiber, a full-field optical coherence
tomography using a home-designed Mirau objective realized high quality
images of in vivo and excised skin tissues. With a 40 × silicone-oil-
immersion Mirau objective, the achieved spatial resolutions in axial and
lateral directions were 0.9 and 0.51 μm, respectively. Such a high spatial
resolution enables the separation of lamellar structure of the full epidermis
in both the cross-sectional and en face planes. The number of layers of
stratum corneum and its thickness were quantitatively measured. This label
free and non-invasive optical probe could be useful for evaluating the water
barrier of skin tissue in clinics. As a preliminary in vivo experiment, the
blood vessel in dermis was also observed, and the flowing of the red blood
cells and location of the melanocyte were traced.
©2014 Optical Society of America
OCIS codes: (060.2380) Fiber optics sources and detectors; (170.4500) Optical coherence
tomography; (160.1435) Biomaterials; (170.3880) Medical and biological imaging; (180.3170)
Interference microscopy.

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1. Introduction
In recent years, optical coherence tomography (OCT) has been widely applied on three-
dimensional (3-D) image reconstruction of skin tissue [1,2]. In epidermis, to non-invasively
probe the layer parameters (LPs), such as average total thickness (a-TT), average number of
layers (a-NOLs), and average cellular layer thickness (a-CLT), for stratum corneum (SC)
becomes important for evaluating the skin moisturization of epidermis [3]. On this
application, axial resolution better than 1.2 μm in tissue is the doorsill to measure LPs of the
SC [4]. Besides, the morphology of single 3-D epidermal cell is also important for early
detection of normal and abnormal cells of pre-cancer diagnosis [5]. These all require sub-
micron spatial resolution in tissue [6]. Full-field OCT (FF-OCT) [7,8] utilizing two-
dimensional CCD/CMOS camera has the opportunity to observe the layer structure of SC [9],

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3002
especially for en face monitoring. Typically, the detection sensitivity of FF-OCT using
CCD/CMOS camera is about 80 dB [10], related to the camera area size and en face frame
rate.
Keratinocyte and melanocyte are the two major cell types in epidermis, with a normal size
from 10 to 50 μm [11]. The epidermis can be divided into several layers, which are stratum
basale at the bottom, stratum spinosum, stratum granulosum, stratum lucidum, and SC on the
top, through keratinization process within about one month. In epidermis, melanocytes are
interspersed at stratum basale with stretching dendrites [12]. For skin care aspect, the
proliferation and differentiation of keratinocyte affect the capability of epidermal moisture
lock [13,14] and dry skin disease [15].

Fig. 1. (a) The cross-sectional image (natural logarithmic gray level, 8-bit filtered by Image J)
of excised buttock (55-year-old, female) and (b) the corresponding anatomical sketch of skin
tissue. In between (a) and (b) shows the corresponding layers. The white arrow indicates the
nucleus of stratum spinosum. (c) shows the in vivo cross-sectional image of the forearm skin
(35-year-old, male), where yellow and blue arrows indicate the dermis-epidermis junction and
blood vessel, respectively. The green arrow heads mark the boundaries of SC. In (a), the SC is
much thicker than that of (c) because of z-axial expansion induced by water hydration. In (c),
58% glycerin was used as the index-matching liquid between in vivo human skin and CG. In
(a)-(c), red arrows are the boundaries between CGs and index-matching liquids. (d) shows the
en face image of (c) at a depth of 46 μm (position of pink dash-dot line in (c)). In (d), the
purple arrows point to the melanocyte along its dendrites, traced from melanin caps of the
shallower en face images. The white spots in (c) and (d) pointed by orange arrows are the
melanin caps. Media 1 and Media 2 respectively show the positional scans of cross-sectional
and en face planes correspondingly for (c) and (d) from a 3-D image stack. (e) shows the
oblique view of 3-D image of in vivo human skin. The scale bars are all 15 μm. The incident
power onto the sample and the CCD exposure time from 3-D stack are 5 mW and 210 μs. To
compare (a) with (c), in vivo skin tissue can provide active morphological information, like
exact LPs of SC, melanin caps, and dynamically flowing of red blood cells (see Media 2).

Typically, LPs of the SC in broad area are related to skin barrier function. In addition to
OCT methods, a-TT of the SC was indirectly verified via z-axial population of water content
by confocal Raman spectroscopy [13,16] or was directly measured by multiphoton laser
tomography [17,18]. For confocal Raman spectroscopy and confocal reflectance microscope
[16], they both need the database of a-NOLs from frozen section [19] to verify the a-CLT of
SC. For multiphoton laser tomography, such as nonlinear effect (i.e. second/third harmonic

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3003
generation and coherent anti-Stokes Raman spectroscopy), it needs high power density (about
30-100 mW average power, with transient focal spot size less than 0.5-μm-diameter) and long
measurement time (about 15-30 minutes for 100-μm depth) to establish one full 3-D image
stack. Figure 1(a) shows the cross-sectional image from a 3-D en face stack scanned by the
Mirau-based FF-OCT with a 40 × home-designed Mirau objective; whereas, Fig. 1(b)
represents the schematic cross-sectional structure of an anatomical illustration. Via the cross-
sectional images at different positions (see Media 1), layer-to-layer boundaries become easy
to observe. Most of the skin does not have stratum lucidum, except for palm and sole. Figures
1(c) and 1(d) are the in vivo images of human forearm skin in cross-sectional and en face
planes, respectively. To compare Figs. 1(a) and 1(c), melanin caps of Fig. 1(a) disappeared
because the excised skin tissue is gradually denatured after skin tissue was immersed in
phosphate-buffered saline (PBS). Figure 1(e) represents the oblique view of 3-D image,
which is the same in vivo tissue of Fig. 1(c) and 1(d). The incident power and 3-D imaging
time (about 100-μm-depth) of this system were 5 mW (focal spot size about 220-μm-
diameter) and 2 minutes, respectively. The scan speed of en face images is 4.3 frame/sec.
Compared with the single-point scanning via Ce3+:YAG double-clad crystal fiber light source
[20,21], this platform provides high frame speed and low incident power for 3-D
reconstruction of skin tissue.
In this study, a Ce3+:YAG single-clad crystal fiber (SCF), drawn by laser-heated pedestal
growth [22] and cladded by borosilicate glass [23], provides high brightness and high
numerical aperture (NA) output light. The diameters of core and cladding are correspondingly
90 and 330 μm. The broadband output power after coupling into multi-mode fiber (Thorlabs,
400-μm-diameter, NA: 0.39, America) is 30 mW, and becomes 5 mW after passing through
Mirau objective onto the sample. This light source is eminently suitable for FF-OCT because
it is unnecessary to use infrared-cut filter to prevent from thermal problem on sample [24].
With a Gaussian-like broadband spectrum [20,21] the ghost image effect coming from side-
band noise is suppressed. Circularly polarized incident light was adopted because it can
provide deeper penetration [25]; meanwhile, weak signal light can be strongly enhanced by
the reference one.
2. Methods of Mirau-based FF-OCT
2-1 Experimental setup and system performance
Figure 2(a) shows the schematic diagram of Mirau-based FF-OCT. The Ce3+:YAG SCF was
pumped by a 1-W, 445-nm laser diode (Nichia, #NDB7875, Japan) through collimating and
focusing module, including a 60 × aspheric lens, a band-wave-pass filter (Semrock, #FF01-
445/45, America), and a 40 × achromatic lens, where the function of band-wave-pass filter is
to reflect the backward broadband light back to the SCF, to enhance the total output power.
The broadband light emerging from the output terminal of the SCF was coupling into multi-
mode fiber and was then collimated by an objective lens, where the center wavelength and
bandwidth of light after SCF are respectively 560 and 95 nm. Then, this light reflected by
broadband polarizing cubic beamsplitter became vertically polarized. After achromatic
quarter wave plate, it changed to circular polarization. The circularly polarized light became
counter circular polarization when reflected back from reference and sample arms through
Mirau objective. Finally, light beams from reference and sample arms both became
horizontally polarized after passing achromatic quarter wave plate again (see green arrows in
Fig. 2(a)). As a result, the back-reflected light beams from sample and reference arms were
combined after broadband polarizing cubic beamsplitter, reflected by mirror, and then
projected onto CCD (Imperx, #ICL-B0620, 640 × 480 pixels, America), to generate the
interferometric signal with a frame rate of 260 frame/s During one period of interferometric
carrier signal, there are 60 sampling frames.
As shown in Fig. 2(b), the home-designed silicone-oil-immersion Mirau objective is
composed of a water-immersion objective lens (Olympus, LUMPLFLN 40 × W, NA: 0.8,
field-of-view: 550 μm, Japan), a ring holder, two fused silica glass plates (thickness: 150 μm,

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3004
λ/10 flatness). The diameter of focal field in water is about 220 μm (1/3 field-of-view was
used). The Mirau objective was fixed on a z-axial piezo-electric transducer (PZT) (PI, #P-
720, Germany). Cover glass (see Fig. 2(a)), the same thickness as fused silica glass plates (see
Fig. 2(b)), was laminated under the sample. The total travelling range of the PZT with open-
loop control is 112 μm. A 500-μm-diameter black ink absorber is used, to well-match the
index of first glass plate and to absorb the stray light back to the CCD, for eliminating the DC
term of intensity. After coating the interlaced layers by TiO2/SiO2, T/R = 60/40 (T:
transmittance; R: reflectance; noil = 1.406) broadband beamsplitter coating was coated on the
top of second glass plate (GP2). The reflection coating (RC) under first glass plate (GP1) is
about 4% as noil = 1.406.

Fig. 2. (a) Experimental setup of the Mirau-based FF-OCT. LD: 445-nm laser diode; CM1 and
CM2: collimating and focusing modules; SCF: single-clad crystal fiber; MMF: multi-mode
fiber; L1: 20 × objective lens (NA: 0.4); LWPF: optical long-wave-pass filter; BPCB:
broadband polarizing cubic beamsplitter; M: mirror; AQWP: achromatic quarter wave plate;
PZT: piezo-electric transducer; L2: home-designed Mirau objective; S: sample; CG: cover
glass; LS: transversally moved linear stage; L3: tube lens (focal length: 15 cm); CCD: charge-
coupled device camera. In (a), green arrows show the polarization states. (b) The schematic
illustration of L2. OL: 40 × silicone-oil-immersion objective; GP1 and GP2: first and second
glass plates; BBC: broadband beamsplitter coating; RC: reflection coating; RH: ring holder; B:
500-μm-diameter black absorber (n = 1.48); nwater and noil: refractive indices of water and
silicone oil. (c) The emission spectrum of Ce3+:YAG SCF, where the inset shows the end view
of SCF. (d) and (e) show the optical path difference and the lateral scanning in water, which
reveal the axial and transversal resolutions in water are respectively 0.91 and 0.56 μm. The
inset of (e) is a straight broken glass plate imaged by the FF-OCT, to find the transversal
resolution, where the interval between two red solid circles is 0.225 μm (CCD pixel
resolution).

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3005
 The PZT was biased by an amplified signal from a DAQ card (NI, #PCI-4461, America)
with an open-loop mode. Z-axial position of the PZT versus input voltage was recorded by
the counted wave numbers and phase difference of a He-Ne laser via Michelson
interferometer. So, the hysteretic movement of the PZT was experimentally compensated via
recorded position versus voltage curve. Figure 2(c) shows the emission spectrum of a
Ce3+:YAG SCF. The interferometric signal intensity of A-scan reflected from the boundary
between water and glass plate was measured by one pixel of CCD (see Fig. 2(d)). The
intensity of carrier envelope from carrier signal in Fig. 2(d) was calculated after band-pass
filter and Hilbert transform. Following the calculation of literature [7,9], the detection
sensitivity is about 81 dB. Referring to the literature [20], the noise floor of this system is
substantially suppressed by stronger confocal gate (NA: 0.8) effect, and then the effect of
ghost image is further leveled down. The home-designed silicone-oil-immersion Mirau
objective provides experimental resolutions of Ra = 0.91 μm (see Fig. 2(d)) and Rt = 0.56 μm
(see Fig. 2(e)) along axial and transversal directions at the surface of water medium (or Ra =
0.90 μm and Rt = 0.51 μm at the surface of SC (n = 1.47 after water hydration)), respectively
[26]; whereas, the theoretical spatial resolutions at the surface of water following diffraction
limits are Ra = 0.56 μm and Rt = 0.43 μm (or Ra = 0.55 μm and Rt = 0.39 μm at the surface of
SC) according to Eq. (1) [7].
−1
 nwater nsample 
Δzeff =  +  , (1)
 nsample Δzconfocal nwater Δzcoherence 
where Δzeff means the effective axial resolution contributed by Δzconfocal (confocal gate in
water, equal to λ0nwater/NA2, about 1.16 μm for 40 × silicone-oil-immersion objective (NA:
0.8)) and Δzcoherence (coherent gate in water, equal to 0.44λ02/nwaterΔλ, about 1.09 μm for
Ce3+:YAG light source with the same objective). nsample and nwater are the refractive indices of
the sample and the water, respectively. λ0 and Δλ are the central wavelength and the
bandwidth, of the light source. In Fig. 2(b), 40 × OL is used for water-immersion. As the
spaces are injected into silicone oil, Ra and Rt become worse. So, experimental resolution is
not as good as theoretical one.
2-2 Signal processing
Typically, FF-OCT takes the en face image from calculating the stack information via phase-
stepped technique with single-shot CCD at 0°, 90°, 180°, and 270° [27], of which the phase
was shifted by triangularly oscillated motion of PZT. As the exposed time of one en face
image increases, the detection sensitivity becomes better. Then, 3-D image is reconstructed
by piling up the en face images along z-axis. Different from classical FF-OCT, the Mirau-
based FF-OCT in this study reconstructs the 3-D image stack via sequential interferometric
signals.
Assuming a sinusoidal interferometric signal with N sampling points during each carrier,
the intensity envelope I(x,y,z0)env at a depth of z0 during one carrier can be approximately
expressed as
1/ 2
2P  N / P 2
I ( x, y, z0 )env =   [ I i ( x, y ) − I j ( x, y )]  , (2)
N i > j =1 
where z0 is the central position of the calculated single carrier wave. (x,y) is the pixel location
on the CCD. N/P is the number of calculating intervals. Ii(x,y) and Ij(x,y) are two P-times-
averaged intensities of the N/P intervals. The calculated result of Eq. (1) is insensitive of the
phase of the single carrier wave, where N/P  3 must be limited. Equation (2) can be applied
on this continuous scanning FF-OCT without any Hilbert transform and further band-pass
filter to detect the envelope of interferometric carrier wave. In addition, Eq. (2) is a

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3006
normalized formula for calculating the signal envelope intensity of a sequential carrier.
Taking N/P = 3 as a example, Eq. (2) becomes

2
I ( x , y , z0 ) env = {[ I 2 ( x , y ) − I1 ( x , y )] + [ I 3 ( x , y ) − I1 ( x , y )] + [ I 3 ( x, y ) − I 2 ( x , y )] } . (3)
2 2 2 1/ 2

3
Equation (3) is consistent with the Eq. (4) in [28], and this equation was also commonly used
in fluorescence microscopy for background subtraction [29].

Fig. 3. (a) The magnified carrier signal measured by the Mirau-based FF-OCT. In (a), red and
green markers are respectively the averaged intensities of N/P = 3 and 4; whereas, red and
greed lines depict the interval separations. (b) The calculated envelopes in linear scale using
Eq. (2) by N/P = 3, 4, 39, 156, and Hilbert transform after using a band-pass filter,
respectively. The inset shows the envelopes in logarithmic scale, where the noise floors of N/P
= 3, 4, and the sequential raw signal with Hilbert transform after the band-pass filter are very
close.

In most applications, the amplitude of sinusoidal wave is amplitude-modulated. Figure

3(a) shows the magnified experimental carrier signal scanned by Mirau-based FF-OCT. As
the sinusoidal wave is Gaussian-modulated, the calculated envelopes of N/P = 3, 4, 39, and
156 are calculated in Fig. 3(b) according to Eq. (2). Peak value of N/P = 156 is the nearest
result when compared to the raw data using Hilbert transform with band-pass filter; whereas,
N/P = 3 has the lowest noise floor. In Fig. 3, the percentages of peak values of N/P = 3, 4, 39,
and 156 versus the peak value of raw data using Hilbert transform with band-pass filter are
correspondingly 82.7%, 89.0%, 99.3%, and 99.5%. In Fig. 3, carrier signal was measured
under lower moving speed of open-loop PZT, and there are 156 sampling point during one
period of interferometric carrier signal. For logarithmic image, N/P = 3 has best noise floor,
and the calculating process is also much faster than that of N/P = 156. But, N/P = 3 has poor
result of peak value. To sum up, N/P = 4 has better benefit for en face image calculation from
stack signal. In the experiment, N/P = 4 was used to reconstruct the 3-D morphology of SC.

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3007
3. Experimental results and discussions

(a) (b)

(c) (d)

(e)

Fig. 4. Cross-sectional images of an excised tissue from buttock (54-year-old, female). (a) and
(b) are the single-trip en face (depth = 60 μm) and cross-sectional (y = 80 μm) 8-bit-depth
images (with nature logarithm scale processed by ImageJ) at a 3-D volume measurement time
of 2 minutes. (c) and (d) are images at the same positions of (a) and (b) with an average after
10 scans. Media 3 shows the en face variances at different depths of (c). The incident powers
on sample and CCD exposure time of (a)-(d) are 460 μW and 3.1 ms. In (d), yellow arrows
indicate the basal cells and blue arrow points out the microvessel. The physical interval
between two en face images is about 0.19 μm. (a) and (c) are the en face images respectively
located at the positions of pink dash-dot lines correspondingly in (b) and (d). In (a)-(d), all the
scale bars are 20 μm. (e) The intensity profiles (gray and red curves) in 10log scale along gray
and red dash-dot arrows of (b) and (d), respectively. After 10 averages, the noise level is
improved by 2.15 dB, which is less than the theoretical estimation of 3.16 dB.

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3008
Figures 4(a) and 4(b) show the en face and cross-sectional images from 3-D en face image
stack; whereas, Figs. 4(c) and 4(d) represent the 10-time-average results of Figs. 4(a) and
4(b). In order to compare the performance after 10-time-average, two axially scanned
intensity profiles of Figs. 4(b) and 4(d) at the same position are plotted together in Fig. 4(e).
The noise level after 10-time-average was improved by 2.15 dB.
For the whole epidermis (refer to Fig. 4(d)), the signals from other layers will affect the
calculation of LPs in SC. In order to directly measure the 3-D LPs of SC, the tested SC was
isolated by chemical and enzymatic digestion techniques via 0.1% trypsin at 37°C for 2 hours
in an incubator [30]. This SC was immersed in 4% formalin to maintain the structure and was
slightly laid on cover glass as shown in Fig. 2(a). Figures 5(a)-5(d) show the cross-sectional
images of the isolated SC at y = 45, 56.25, 67.5, and 78.75 μm. In Fig. 5(e), axial LPs of the
SC from Fig. 5(a) were measured and then calculated.

Fig. 5. Images of isolated SC from excised buttock sample. (a)-(d) are the cross-sectional
images in x-z planes at the corresponding positions of y = 45, 56.25, 67.5, and 78.75 μm from
3-D en face stack. Red and yellow arrows respectively refer to the wrinkles and the hollow
cavities of the sample. The scale bars are all 15 μm. (e) The cross-line profile of (a) along
white dash-dot arrow, where red points and blue dash line are separately peak values and set
threshold of the intensity profile. The interval between two nearest peaks means a CLT. The
number of CLT is NOL. The interval between first and last peaks means TT. Ith is the intensity
threshold between signal spikes and noise floor.

Table 1 shows the regional LPs of the SC from Fig. 5. Better than the results from single
2-dimensional image, 3-D image of SC after trypsin can provide more significant LPs of the
SC, to evaluate the skin water barrier. Typically, SC immersed in water or PBS after one hour

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3009
generates about 40% z-axial expansion from hydration [12]. The 3-D measurements of a-TT
and a-NOLs from buttock with deducting hydration are 14.56 ± 2.38 μm and 11.72 ± 3.00,
respectively. In the literature, a-TT and a-NOLs of the SC at buttock using frozen section are
respectively 14.9 ± 3.4 μm (m = 61) [31] and 12 ± 4 (m = 20) [12,22], where m means the
number of samples. Actually, histological biopsy is difficult to provide 3-D information of
SC. Comparing Figs. 5(a)-5(d) with Table 1, hollow cavities increase the standard deviation
of a-CLT. In Table 1, mean value of Fig. 5(a)-5(d) is very close to the result of 3-D vision. In
other words, sampling method (mean value of Fig. 5(a)-5(d)) of 3-D information is sufficient
enough to be used for evaluating significant LPs of the SC.
In this study, a desktop system was established [32], which is suitable to take the 3-D skin
images of forearm and face for whole epidermis. As deeper information is necessary to
image, like collagen, Mirau objective has to be changed into low magnification one. Full-
depth epidermis images of skin tissues for excised and in vivo cases were both reconstructed
in detail via the Mirau-based FF-OCT. In Media 2 (in vivo skin tissue), flowing of red blood
cells was observed, but the flow speed of red blood cells still cannot be estimated. It becomes
capable as the en face frame rate is improved. In contrast, Media 3 (excised skin tissue) did
not represent any flowing imprint. In Fig. 1(d), the melanocyte was traced by its melanosome-
filled dendrites. It needs to catch the timing that the melanocyte is secreting and transporting
the melanosome via dendrites. In this experiment, LPs of the isolated SC were measured by
Mirau-based OCT, but the concrete value of this measuring method is for in vivo skin. In vivo
skin cannot use trypsin to isolate the SC away, so it is necessary to build image segmentation
as boundary between SC and stratum granulosum, to take the in vivo LPs from patients
directly. This information will be very helpful for the judgments of water barrier (cosmetics)
and hyperkeratosis (skin disease) [33].
Table 1. LPs with standard deviations for the isolated SC from Fig. 5 with deducting
40% water hydration.
a-TT (μm) a-NOLs a-CLT (μm)
Sampling area
(Average ± SD)
Figure 5(a) 14.17 ± 1.42 12.02 ± 2.52 1.19 ± 0.60
Figure 5(b) 15.69 ± 2.73 12.32 ± 3.53 1.29 ± 0.94
Figure 5(c) 15.77 ± 2.40 12.69 ± 3.15 1.25 ± 1.02
Figure 5(d) 14.00 ± 2.66 11.30 ± 2.93 1.27 ± 0.75
Mean valuea 14.91 ± 2.30 12.08 ± 3.03 1.25 ± 0.83
3-D visionb 14.56 ± 2.38 11.72 ± 3.00 1.27 ± 0.84
a
Mean value is the average of Figs. 5(a)-5(d).
b
3-D vision means all the 640 × 480 A-lines from 3-D en face stack were calculated.

4. Conclusions
A Mirau-based FF-OCT was demonstrated by reconstructing the 3-D skin tissue and
measuring the LPs of the prepared SC. It took about 2 minutes to scan and reconstruct full
cuboidal image stack of tissue with 144 (x-axis) × 108 (y-axis) × 100 (z-axis) μm3 using less
than 5 mW incident power. For in vivo skin tissue, 3-D skin morphology was also visualized,
but melanin caps will absorb most of the visible light from Ce3+:YAG SCF. The differences
between in vivo and excised skin tissues coming from different locations, such as hydration of
SC, denature of melanin caps, and information of red blood cells, were verified. In the future,
this system can directly applied on the clinical observation of deeper position, as the light
source is changed to Ti:sapphire SCF broadband light source. Based on this system, the a-TT
and a-NOLs from excised buttock with deducting hydration were measured. After the
analysis of more patients, it is capable to define the skin ages of water barrier. This is very
important to the medical and cosmetic areas of dermatology.
Acknowledgments
This work is partially supported by National Science Council, Taiwan, through the project of
NSC 100-3113-P-002-008.

#213397 - $15.00 USD Received 9 Jun 2014; revised 15 Jul 2014; accepted 5 Aug 2014; published 8 Aug 2014
(C) 2014 OSA 1 September 2014 | Vol. 5, No. 9 | DOI:10.1364/BOE.5.003001 | BIOMEDICAL OPTICS EXPRESS 3010