The value is calculated by dividing the length of the recovered part 'B' by the length of the entire wound 'A', (B2) The results show a quantitative analysis of the regenerated wound area in the engineered biopsy skin using the 3D-based segmentation algorithm Images of October. Cross-sectional (2D) and 3D OCT image before and after chitosan-based tissue adhesive treatment.
Introduction
Research Background
Among these techniques, confocal microscopy and multiphoton microscopy have been widely used to obtain various information of biological tissues with a chemical composition such as fluorescent markers. Multiphoton microscopy, which is a nonlinear optical imaging mode to overcome the diffraction limit, enables long-term imaging without photo-bleaching and photo-damage phenomena and high signal-to-noise ratio with a high penetration depth of up to 500 μm compared to confocal microscopy. [11].
Research Purpose and Motivation
The confocal microscopy used in various applications for evaluation of normal or abnormal tissues because it provides a high-resolution image of ex vivo and in vivo tissues for morphological and structural investigation with a penetration depth of about 200μm [9, 10] . Unlike the confocal microscopy that used the pinhole to block focused light, the multiphoton microscopy acquired the signals based on multiphoton absorption to reduce optical attenuation and background noise by multiple photons of longer wavelengths.
Optical Coherence Tomography for Wound Healing
Optical Coherence Tomography (OCT)
- Summary of OCT
- OCT system specification
The probe arm responsible for imaging the skin sample was composed of a collimator, two-axis galvanometer scanner, and objective lens providing a lateral resolution of ~15 μm in tissue. Reflected light from the sample and reference arm was combined and these interference signals were converted into an electrical signal using a balanced photodetector (PDB450C, Thorlabs Inc., USA).
OCT Monitoring in In Vivo and In Vitro Skin Model
- Engineered Skin Fabrication
- Quantitative Analysis of Laser-treated Engineered Skin
- Monitoring of Epidermal Regeneration in Wound Engineered Skin
- Quantification of Wound Regeneration in Rodents Wound Model
In Figure 2-16(B), we obtained an OCT image of a wound in a rat model with a skin incision after treatment with a chitosan-based tissue adhesive. A chitosan-based tissue adhesive was applied to the wound site of the skin incision model using a customized syringe.
Serial Block-face Optical Coherence Microscopy for Whole Tissue Imaging
Optical Coherence Microscopy (OCM)
- Hardware Configuration and Specification
- System Evaluation: Label-free Visualization of Hair Follicle
Skin tissue samples were prepared immediately after waxing for an area of 5 mm × 5 mm and after hair follicle growth for two weeks. Immediately after hair removal, we found that traces of hair follicles remained in the superficial layer of the skin and we confirmed that OCM provides excellent contrast for different structures in the skin. The images shown in the second bottom section represent the development of hair and hair follicles two weeks after hair removal.
We also observed the detailed structure of hair and follicles in OCM en-face images, such as hair bulb, hair root, hair papillae, and the hair follicle wall consisted of the epidermal and dermal sheath. The growth and maturation process of hair and follicles was observed on the date. In figure 3-4, we acquired OCM en-face image of the hair follicle development for 2 weeks after hair growth.
The OCM image immediately after waxing shows traces of hair follicles on the superficial layer of the skin.
Depth Trajectory Tracking OCM
- Principle of Depth Trajectory Tracking Technique
- High-quality Image Acquisition in Tilted and Uneven Tissue Surface
In the first scan by OCM, we used a low magnification objective lens that has a large field of view and wide depth-of-focus to obtain height information between the surfaces of the sample. In the first step, OCM scanned the sample with a low magnification objective lens that has a large field of view and wide depth-of-focus to obtain topological data and height information between the surfaces of the sample. The low magnification objective lens allowed to obtain larger diameter window of the optical signals used to find the height changes on the sample surface.
Low magnification is also suitable for prescanning to obtain the height information because the wide depth of focus contributed to less signal reduction due to tilt of tiled sample and curvature of the surface. In Figure 3-5(B) of the second scan, the topology map obtained in the first scan corresponds to the approximate slope over the area of interest where it is used for lookup table for an axial position. With limited transverse resolution, morphological features of the kidney were not identified in the 4X en-face OCM image in Figure 3-7(A).
The surface of the sample may not be perfect due to the uneven force applied while cutting the tissue using vibratome.
Serial Block-face OCM for High-resolution Whole Tissue Imaging
- Serial Block-face Imaging Technique: Whole Kidney Imaging with Vessel Segmentation 44
- Protocol for Fully Automated Whole Tissue Imaging
- High Throughput Whole Tissue Imaging for Histopathological Study
In the next step, the OCM en-face image obtained in the top layer of the sample. A three-dimensional image of the entire tissue was obtained by repeating tissue cutting and OCM imaging. By imaging the whole tissue of the mouse kidney, we were able to identify the internal structure of the kidney in different directions.
In the whole tissue image of the brain in Figure 3.17, we identified a clear high-resolution tissue structure in each coronal, sagittal and transverse direction of the three-dimensional brain image. The three-dimensional image of the spine shows the clear structure and orientation of the neural fiber. In the three-dimensional reconstructed heart image, we confirmed the clear structure of the heart, as well as tissue morphologies such as.
Projection images and three-dimensional vascular images of blood vessels in the liver tissue are obtained, allowing quantitative analysis of the blood vessels.
Hand-held Probe based Portable OCT System for Human Target Study
Hand-held Probe based portable OCT System
- Development of Hand-held Probe
- Portable OCT System with Hand-held Probe
For the development of the portable probe, it is very important to miniaturize the dimensions of the portable probes while maintaining the same performance of the benchtop image acquisition arm. The portable grip-type probe, which will be used as a sample arm for acquiring OCT images, was fabricated by using a MEMS scanner instead of the galvanometer scanner of the existing OCT imaging system for miniaturization. The portable handgrip type probe is designed to enable stable and comfortable image capture when the user approaches a target using a probe. (B) Prototype wearable probe with 3D printing, (C) Final assembled handle-type wearable probe.
And then we collected the user's opinions and modified the design to produce the final product of the grip type hand probe. A), (B) Sketch and 3D CAD drawing of the early concepts of handheld OCT system with hand probe. We have developed a LabVIEW-based software for the operation of the handheld-based portable OCT system.
We have performed clinical studies on the human body using a complete portable OCT system based on a hand probe and performed various quantitative analyzes especially on human skin.
Dynamic Monitoring and Quantification of Human Skin Conditions
- Applications for Human Skin Monitoring
- Quantitative Evaluation of Human Skin Wrinkle Roughness
Quantitative analysis of light transmission into human skin after treatment with powder-like cosmetics. We performed a quantitative analysis of the depth and volume of skin wrinkles based on these image datasets. Then, the area between the skin surface line and the estimated surface line was extracted, and the wrinkle depth and volume of the human skin were calculated using this area.
Also, the 3D reconstructed skin wrinkles were confirmed to correlate with the 3D OCT image of the skin. PRIMOS is a non-invasive, rapid and direct measurement of skin surface area with high accuracy [66]. Through this experiment, the influence of the aging process on the roughness values of the skin surface was confirmed.
OCT visualized the morphological variation of the skin surface in a three-dimensional and high-resolution approach.
Imaging Guided Three-dimensional Modeling for Tissue Fabrication
Wrinkle Mimicked Engineered Skin Model
- Fabrication Protocol for Winkle Mimicked Engineered Skin
- Evaluation of Wrinkled Engineered Skin Model
In Figure 5-2, we fabricated the wrinkle pattern mold to develop the wrinkle-mimicking engineered skin model using a 3D printing technique. The tissue culture process of engineered skin fabricated by the traditional method as a control and the eye mimicked the engineered skin using the eye model mold. The fabrication process of the wrinkled engineered skin was carried out in the order of (1) wrinkle pattern formation on the skin layer, (2) cultivation of the skin layer, and (3) cultivation of the epidermis layer.
Wrinkle-mimicking engineered skin was then created by forming epidermal layers by incubating in air exposure for 14 days. We could visually observe the pattern of wrinkles made by the 3D printed mold on the fully fabricated engineered skin. The fully cultured wrinkle mimicked the engineered skin created by different eye pattern gaps in the eye pattern mold produced by the 3D printer.
Evaluation of the winkle-mimicking engineered skin using the H&E staining compared to the native engineered skin without winkle patterning.
OCT Imaging Guided Engineered Cornea Fabrication
- Fabrication Protocol for Personalized Engineered Cornea
- Personalized Engineered Cornea for Corneal Transplantation
The 3D structural information was reverse engineered to create a 3D printed mold to fabricate the engineered cornea. In Figure 5-11, we reconstructed the 3D structure based on the region segmentation results for the regions to be replaced with the engineered cornea. The 3D structural information was reverse engineered to create a 3D printed mold using a 3D printer to produce the engineered cornea.
The engineered cornea made with different material candidates that have similar mechanical and optical properties to the native cornea. Evaluation of candidate materials for personalized engineered cornea in corneal transplantation using the 2D and 3D-OCT images. Here, we proposed imaging-guided tissue fabrication protocol for personalized engineered cornea, which mimicked structural properties of native cornea.
The 3D printed mold is designed for manufacturing engineered cornea based on personalized factors such as thickness and curvature.
Discussions and Conclusions
Discussions and Conclusions
For the evaluation of the histopathological analysis using OCM, we observed the development of hair follicles and performed a comparison study with the tissue staining method. Therefore, we introduced depth trajectory tracking technique to acquire OCM images of homogeneous quality regardless of the height difference of the sample surface. The SB-OCM system was fabricated in the automated system that performs large field of view optical imaging and physical tissue sectioning of the agarose-embedded tissue.
The handheld probe can be easily accessed by users and image acquisition is possible regardless of the shape or position of the target. However, most of the processes from the mold pattern shape to the engineered skin culture were related to the formation of wrinkles and curvature, which required very careful adjustment. We also attempted to fabricate the engineered cornea to mimic the structural, physical, and optical characteristics of the native cornea, as well as its structure.
We reconstructed the 3D structure of the surgical area to be peeled using the image processing based on previously obtained information.
Future Perspectives
Coulman, S.A., et al., In vivo, in situ imaging of microneedle insertion into the skin of human subjects using optical coherence tomography. Jung, W., et al., Optical coherence tomography for in vitro monitoring of wound healing after laser irradiation. Yeh, A.T., et al., Imaging wound healing using optical coherence tomography and multiphoton microscopy in an in vitro skin equivalent tissue model.
Tsai, M.-T., et al., Monitoring wound healing process of human skin after fractional laser treatments with optical coherence tomography. Banzhaf, CA, et al., Spatial closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy. Ramirez, D.M., et al., Serial multiphoton tomography and analysis of volumetric images of the mouse brain, in Multiphoton Microscopy.
Sakai, S., et al., In vivo evaluation of human skin anisotropy by polarization-sensitive optical coherence tomography.
