Int J Burn Trauma 2018;8(2):17-25 www.IJBT.org /ISSN:2160-2026/IJBT0075736 Review Article Application of laser scanning confocal microscopy in the soft tissue exquisite structure for 3D scan Zhaoqiang Zhang1, Mohamed Ibrahim2, Yang Fu3, Xujia Wu3, Fei Ren4, Lei Chen5 1Department of Oral and Maxillofacial Surgery, Stomatological Hospital, Southern Medical University, No. 366, South of Jiangnan Road, Guangzhou 510280, Guangdong, P. R. China; 2Division of Plastic and Reconstructive Surgery, Department of Surgery, Duke University Medical Center, Box 3181, Durham 27710, North Carolina, USA; 3Department of Clinical Medicine, Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou 510080, Guangdong, P. R. China; 4Department of Oral Medicine, Stomatological Hospital, Southern Medical University at Haizhu Square, No. 180, Taikang Road, Guangzhou 510280, Guangdong, P. R. China; 5Department of Burns, The 1st Affiliated Hospital of Sun Yat-sen University, 58 2nd Zhongshan Road, Guangzhou 510080, Guangdong, P. R. China Received December 25, 2017; Accepted March 23, 2018; Epub April 5, 2018; Published April 15, 2018 Abstract: Three-dimensional (3D) printing is a new developing technology for printing individualized materials swiftly and precisely in the field of biological medicine (especially tissue-engineered materials). Prior to printing, it is nec- essary to scan the structure of the natural biological tissue, then construct the 3D printing digital model through optimizing the scanned data. By searching the literatures, magazines at home and abroad, this article reviewed the current status, main processes and matters needing attention of confocal laser scanning microscope (LSCM) in the application of soft tissue fine structure 3D scanning, empathizing the significance of LSCM in this field. Keywords: LSCM, 3D printing, tissue engineering Introduction because of the similarity of the tissue structure to that of human, which are of most concern to As an important component of tissue engineer- people. However, due to the limitation of work- ing research [1-3], the structure and composi- manship, swine and bovine acellular matrices tion of scaffold material have important influ- have certain immunogenicity with poor nutri- ences on growth and function of seeded cells, tion, penetration of material, slow cellulariza- which is one of the key factors of the success tion and vascularization as well as low survival of tissue-engineered material. What an ideal rates, hence can only be used for provisional scaffold material is it should be biocompatible, wound covering. As a result, researchers start- biodegradable and mechanically appropriate. ed to shift their focus to collagen [9, 10], an However, the present synthetic degradable essential structural protein in dermal matrix’s scaffold materials such as polylactic acid and connective tissue. Taking an advantage of the polyglycolic acid fails to provide a good micro- flexible properties of collagen, they hoped to environment with functions such as growth, reconstruct a scaffold material that encourag- proliferation, interchange of material and sig- es cell growth and rapid vascularization with naling stimuli for cell repair like a natural scaf- the help of technologies such as freeze-drying. fold material (acellular matrix tissue). Besides, with the advancement of organ transplanta- It is challenging for traditional preparation tion, the source of allogeneic natural scaffold methods of scaffolds for tissue engineering material is limited, carried with a risk of disease to overcome problems such as material cell transmission (such as AIDS). That’s why hetero- support and vascularization. Also there are no geneous acellular matrix tissue has attracted personalized characteristics of the structure more attention. obtained by these traditional methods. Thanks to the emergence of 3D printing, it has intro- The sources of allogeneic biologic scaffold duced a new hope to solve this issue. With3D material are usually swine and bovine [4-8], printing technology, an optimized data model LSCM in the soft tissue exquisite structure for 3D scan can be constructed in accordance with charac- Status worldwide teristics of patients’ injured parts. Hence, tis- sue engineer scaffolds can be prepared rapidly In 1985, first research contribution about and accurately based on 3D printing with the LSCM biological application by Wijnaendts Van advantages of realizing close and “seamless” Resandt was noted [29, 30]. Afterwards, the connection with an injured part on the exter- confocal microscope has been widely used in nal structure and matching with defects on the biology and has become a powerful research fine (microcosmic) structure to obtain the best tool in research of cytobiology, morphology, remediation effect. pharmacology and neurosciences, etc. With advancements in science, the confocal micros- The main purpose of the 3D printing in tissue- copy is now not only restricted to the LSCM. engineered material is to obtain accurate 3D In recent years, some foreign scientific research image data of the fine structure of human tis- institutions have invented confocal micro- sue [11, 12]. On one hand, currently, 3D imag- scopes with more perfect functions, such as ing of hard tissues (e.g. bones [13, 14]) have pinhole array disc LSCMs and two-photon made progress rapidly because of existing LSCMs successively. digital medical technology such as computed topography (CT). On the other hand, fine struc- Pinhole array disc LSCM was proposed to solve ture imaging of soft tissues (e.g. skin, nerves problems of confocal detection in the course and vessels) is still limited to 2D. No break- of quick change [31], which was invented by through is noticed until now for 3D imaging of Yokogawa Electric of Japan. This company intro- soft tissues like skin [15-18]. duced twin disc patented technology, including synchronous rotation of a micro-lens array disc In 1957, confocal microscopy technology was and a pinhole array disc. As compared to the proposed [19]. In 1980’s, as an epoch-making conventional laser confocal method that needs high-tech product developed [20-23], LSCM a stage to scan, the pinhole array disc LSCM was a combined outcome of microscope mak- applies CCD as a detector. It is an important ing, photoelectricity, computer technology and tool for in vivo fluorescent imaging of living a modern optical microscope. LSCM 3D stereo cells that only needs synchronous rotation of imaging technology is similar to 3D CT [24, 25], a micro-lens array disc and a pinhole array disc which evenly scans tissues of a specified height to carry out fast confocal detection on objects in certain periods of time, and then overlaps with a maximal full-width collection frame rate images of each scan to build a 3D image. Its of 1000 frame/s [32, 33]. advantages are that it can really reflect distri- bution relations of scaffolds and seed cells by Two-photon LSCM is a new generation of confo- 3D images, especially topological structure in cal microscopes introduced to solve problems scaffolds and cell distribution that can hardly of the photo-bleaching phenomenon of sample be matched by an ordinary optical or electron dye labeling in bioinstrumentation [34]. A sin- microscope [26-28]. gle photon needs high energy to stimulate fluo- rescent molecules. However, under a high pho- Status ton density, two low-energy photons enable to stimulate fluorescence. After get stimulated, History the fluorescent molecule will emit photons of short wavelength and bring same effects. At In 1957, Marvin Minsky firstly founded the con- the same time, low-energy photons reduce focal principle. Later in 1967, Egger and Petran damage (phototoxicity) on samples. The pulse developed the confocal microscopy success- width emitted by this laser is only 100fs with a fully and got an optical section by scanning. In cycle of 80 to 100 MHz, high peak energy and 1977, Sheppard and Wilson put forward Raman low average energy. Also as two-photon excita- spectroscopy for the laser scanner, illuminating tion only occurs on lens focus, the fluorescence the non-linear relationship between light and detection efficiency is able to enhance greatly atoms of illuminated objects. After confocal without a confocal pinhole [35]. microscopes were developed by Biorad (Model SOM-100) and were commercially sold in 1984, Status in China Model MRC-500 applied light beam scanning combined with a bio fluorescent microscope As the trend of using confocal microscopes hereafter in 1986. abroad has increased, China also followed the 18 Int J Burn Trauma 2018;8(2):17-25 LSCM in the soft tissue exquisite structure for 3D scan background noise. However, to make scattered illuminant pass a small hole or to use a laser will form pointolite which will focus on a point of an object, namely the focus. And the plane contains the focus known as the focal plane. According to the principle, a clear 3D image can be ob- tained by moving the lens in directions of x-y-z (Figure 1). Figure 1. After the illuminant passes through the pinhole, only punctiform illuminant emits and focuses on an object via the lens so as to reach a pre- Advantages cise point-to-point effect to scan on plane x-y forming an image of the plane. Deeper scanning can result in more images and the 3D structure drawing As compared to the fluores- can be constructed by computer processing. cence microscopy, following are the advantages of this pace and introduced the first batch of confocal microscope: It can conduct scanning of deep microscopes. So far, many domestic biological tissue and can deal with thick samples; It can research institutions have brought in the confo- remove background interference effectively to cal microscope because of its strong observa- make the image clear and precise; It can collect tion ability in the biology field. The Neurology a series of optical sections; It can carry out Department of the First Affiliated Hospital of dynamic, real-time and non-invasive testing. Sun Yat-Sen University has introduced the two-photon microscope for multiple biological Disadvantages researches.