Numerous methods for the imaging of objects behind scattering media have been proposed [1–3]. One of these techniques is digital holography (DH) , which simultaneously acquires both amplitude and phase information. DH provides three-dimensional (3D) and quantitative phase information, such as the refractive index, by noninvasive and nondestructive measurements. We previously proposed a 3D microscopic imaging method for objects behind scattering media using DH . However, scattering media, such as biological tissues, often comprises multiple scattering layers. Imaging objects behind scattering media is applicable to various fields, and it is considered that if acquiring biological information in vivo becomes possible, it would lead to significant development in the medical field. When reconstructing images of objects behind biological tissue, issues such as shortened optical penetration depth due to scattering and absorption in biological tissues arise. Near-infrared light is known to penetrate biological tissues, like skin, better than visible light . Therefore, in this study, employed nearinfrared light with wavelength of 780 nm to conduct microscopic imaging of an object hidden behind the rat skin. We evaluated the reconstructed image imaged behind the rat skin and compared it to the theoretical resolution.
A schematic diagram of the experimental system is depicted in Fig 1. The beam emitted from the 780-nm laser diode (LD) was split into a reference and an object beam by a polarization beam splitter. These two light waves interfere in-line, and a hologram of the 3D-object is projected as a two-dimensional intensity distribution on the scattering medium. The scattering medium introduces a common random phase φs(r, y) to both the object beam u0(x, y) and the reference beam uR(x, y). Equation (1) describes the intensity l(x, y) of interference fringes,
where u0*(x, y) and uR*(x, y) depict the complex conjugates of u0(x, y) and uR(x, y), respectively. Thus, the random phase φs(x, y) from the scattering medium is canceled. These interference fringes on the scattering media are imaged by the lens, and four interference fringes I(φ) with a relative phase difference φ are captured by the image sensor. The piezoelectric element produced a relative phase difference of π/2, and a complex signal U was calculated using Eq. (2) by four-step phase-shifting .
The in-line DH method allows the exclusion of the spatial carrier frequency for unnecessary image separation and records broad interference fringes. The object image is obtained by calculating the Fresnel diffraction using the complex signal U. Its spatial resolution δx0 is defined by the Rayleigh criterion as follows:
where z0 depicts the optical path length from the object to the scattering medium; D is the diameter of the scattering medium, and λ is the wavelength of the light source. However, when thick scattering media such as living tissues are used instead of a thin scatter plate, interference fringes I(φ) are more affected by the diffusion and absorption of light in the scattering medium as the thickness of the scattering medium increases. To achieve imaging behind a thick scattering media, it is important to select a wavelength that has a high transmittance for the scattering medium.
We used shaved rat-skin tissue as the scattering medium, as shown in Fig. 2(a). The biological skin tissue consists of the epidermis, dermis, and subcutaneous layer over a fascia (Fig. 2(b)) . Under a conventional optical microscope, each layer of the extracted skin sample was observed to contain micro vessels, cells, etc., and it was confirmed that the structure of each layer is different, as shown in Fig. 2(c). The skin sample was fixed between two microscope slides and covered with an aperture of 20 mm. The thickness of the rat skin was determined by averaging five micrometer measurements of the skin sample between the slides five after subtracting the thickness of the microscope slide. The experiment was performed within two hours after excision to simulate the in vivo condition as closely as possible.
Results and Discussion
First, we measured the intensity transmittance of skin using visible and near-infrared light with wavelengths of 632.8 nm and 780 nm. The measured transmittance at 632.8 nm wavelength is 25%, whereas at 780 nm wavelength, it is 41%. At 780 nm, the transmittance was 1.6 times of that at 632.8 nm. We thus experimentally confirmed that nearinfrared light transmits through rat skin to a greater degree and is less affected by scattering compared to visible light.
Followingly, we imaged a TOPPAN test chart behind the scatter plate using near-infrared light at a wavelength of 780 nm. The reconstructed intensity image of Group 4 is shown in Fig. 3(a). The contrast was calculated using the cross-sectional intensity distribution of five vertical strokes that were averaged after extraction of the intensity of each stroke in the vertical direction, as shown by the white frame in Fig. 3(a). In this study, λ = 780 nm, z0 = 50 mm, and D = 20 mm. Using Eq. (3), the theoretical resolution was calculated at 2.38 μm. This value corresponds to the distance between the third and fourth element in Group 4 of the TOPPAN test chart, and its stroke width is d expressed as d = 1/2u, where u is spatial frequency [LP/mm]. The intensity charts are shown in Fig. 3(b) and (c). Five vertical strokes can be identified clearly, including both elements 4 (227 LP/mm) and 5 (250 LP/mm) in the reconstructed image.
Subsequently, we imaged the test chart behind the rat-skin sample of 912μm thickness. The reconstructed intensity image of Group 4 is shown in Fig. 3(d). The intensity charts are shown in Fig. 3(e) and (f). In Fig. 3(f), the five strokes were unclear. Despite the lower contrast of Group 4 shown in Fig. 3(e) compared to that of Fig. 3(b), the five vertical strokes could be confirmed with a pitch that was smaller than the theoretical resolution.
We performed microscopic imaging through scattering media using a near-infrared light source with a wavelength of 780 nm and obtained a favorable reconstructed image. Our results demonstrate that microscopic imaging behind scattering media was possible even when rat skin of about 1-mm-thickness was used as a scattering medium.