Full-depth epidermis tomography using a Mirau-based full-field optical coherence tomography

Abstract

With a Gaussian-like broadband light source from high brightness Ce(3+):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.

Keywords: (060.2380) Fiber optics sources and detectors; (160.1435) Biomaterials; (170.3880) Medical and biological imaging; (170.4500) Optical coherence tomography; (180.3170) Interference microscopy.

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).

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); n_water and n_oil: 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).

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.

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.

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. I_th is the intensity threshold between signal spikes and noise floor.