• Medical Lasers
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• v.10 no.3
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• pp.146-152
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• 2021

Background and Objectives Although lasers have been widely applied in tissue treatment, the light penetration depth in tissues is limited by the tissue turbidity and affected by its absorption and scattering characteristics. This study investigated the effect of using an optical clearing agent (OCA) on tissue to improve the therapeutic effect of 1064 nm wavelength laser light by reducing the heat generated on the skin surface and increasing the penetration depth. Materials and Methods A diode laser (λ = 1064 nm) was applied to a porcine specimen with and without OCA to investigate the penetration depth of the laser light and temperature distribution. A numerical simulation using the finite element method was performed to investigate the temperature distribution of the specimen compared to ex-vivo experiments using a thermocouple and double-integrating sphere to measure the temperature profile and optical properties of the tissue, respectively. Results Simulation results showed a decrease in tissue surface temperature with increased penetration depth when the OCA was applied. Furthermore, both absorption and scattering coefficients decreased with the application of OCA. In ex-vivo experiments, temperatures decreased for the tissue surface and the fat layer with the OCA, but not for the muscle layer. Conclusion The use of an OCA may be helpful for reducing surface heat generation and enhance the light penetration depth in various near-infrared laser treatments.

• Applied Microscopy
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• v.46 no.3
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• pp.134-139
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• 2016

Detailed structural and molecular imaging of intact organs has incurred academic interest because the associated technique is expected to provide innovative information for biological investigation and pathological diagnosis. The conventional methods for volume imaging include reconstruction of images obtained from serially sectioned tissues. This approach requires intense manual work which involves inevitable uncertainty and much time to assemble the whole image of a target organ. Recently, effective tissue clearing techniques including CLARITY and ACT-PRESTO have been reported that enables visualization of molecularly labeled structures within intact organs in three dimensions. The central principle of the methods is transformation of intact tissue into an optically transpicuous and macromolecule permeable state without loss of intrinsic structural integrity. The rapidly evolving protocols enable morphological analysis and molecular labeling of normal and pathological characteristics in large assembled biological systems with single-cell resolution. The deep tissue volume imaging will provide fundamental information about mutual interaction among adjacent structures such as connectivity of neural circuits; meso-connectome and clinically significant structural alterations according to pathologic mechanisms or treatment procedures.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.1-17
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• 2020

Three-dimensional microscopic approaches in histopathology display multiplex properties that present puzzling questions for specimens as related to their comprehensive volumetric information. This information includes spatial distribution of molecules, three-dimensional co-localization, structural formation and whole data set that cannot be determined by two-dimensional section slides due to the inevitable loss of spatial information. Advancement of optical instruments such as two-photon microscopy and high performance objectives with motorized correction collars have narrowed the gap between optical theories and the actual reality of deep tissue imaging. However, the benefits gained by a prolonged working distance, two-photon laser and optimized beam alignment are inevitably diminished because of the light scattering phenomenon that is deeply related to the refractive index mismatch between each cellular component and the surrounding medium. From the first approaches with simple crude refractive index matching techniques to the recent cutting-edge integrated tissue clearing methods, an achievement of transparency without morphological denaturation and eradication of natural and fixation-induced nonspecific autofluorescence out of real signal are key factors to determine the perfection of tissue clearing and the immunofluorescent staining for high contrast images. When performing integrated laboratory workflow of tissue for processing frozen and formalin-fixed tissues, clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue hydrogel (CLARITY), an equipment-based tissue clearing method, is compatible with routine procedures in a histopathology laboratory.

• Molecules and Cells
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• v.39 no.6
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• pp.439-446
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• 2016

Clearing and labeling techniques for large-scale biological tissues enable simultaneous extraction of molecular and structural information with minimal disassembly of the sample, facilitating the integration of molecular, cellular and systems biology across different scales. Recent years have witnessed an explosive increase in the number of such methods and their applications, reflecting heightened interest in organ-wide clearing and labeling across many fields of biology and medicine. In this review, we provide an overview and comparison of existing clearing and labeling techniques and discuss challenges and opportunities in the investigations of large-scale biological systems.

• Journal of the Optical Society of Korea
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• v.17 no.4
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• pp.289-295
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• 2013

Although low-level laser therapy (LLLT) has been a valuable therapeutic technology in the clinic, its efficacy may be reduced in deep tissue layers due to strong light scattering which limits the photon density. In order to enhance the photon density in deep tissue layers, this study developed an optical tissue clearing (OTC) laser probe (OTCLP) system which can utilize four different OTC methods: 1) tissue temperature control from 40 to $10^{\circ}C$; 2) laser pulse frequency from 5 to 30 Hz; 3) glycerol injection at a local region; and 4) a combination of the aforementioned three methods. The efficacy of the OTC methods was evaluated and compared by investigating laser beam profiles in ex-vivo porcine skin samples. Results demonstrated that total (peak) intensity at full width at half maximum of laser beam profile when compared to control data was increased: 1) 1.21(1.39)-fold at $10^{\circ}C$; 2) 1.22 (1.49)-fold at a laser pulse frequency of 5 Hz; 3) 1.64 (2.41)-fold with 95% glycerol injection; 4) 1.86 (3.4)-fold with the combination method. In conclusion, the OTCLP system successfully improved the laser photon density in deep tissue layers and may be utilized as a useful tool in LLLT by increasing laser photon density.

• BMB Reports
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• v.48 no.12
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• pp.655-667
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• 2015

Lineage tracing is a widely used method for understanding cellular dynamics in multicellular organisms during processes such as development, adult tissue maintenance, injury repair and tumorigenesis. Advances in tracing or tracking methods, from light microscopy-based live cell tracking to fluorescent label-tracing with two-photon microscopy, together with emerging tissue clearing strategies and intravital imaging approaches have enabled scientists to decipher adult stem and progenitor cell properties in various tissues and in a wide variety of biological processes. Although technical advances have enabled time-controlled genetic labeling and simultaneous live imaging, a number of obstacles still need to be overcome. In this review, we aim to provide an in-depth description of the traditional use of lineage tracing as well as current strategies and upcoming new methods of labeling and imaging.

• Journal of the Optical Society of Korea
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• v.17 no.1
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• pp.86-90
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• 2013

Various methods have been proposed for enhancing the contrast of laser speckle contrast image (LSCI) in subcutaneous blood flow measurements. However, the LSCI still suffers from low image contrast due to tissue turbidity. Herein, a physicochemical tissue optical clearing (PCTOC) method was employed to enhance the contrast of LSCI. Ex vivo and in vivo experiments were performed with porcine skin samples and male ICR mice, respectively. The ex vivo LSCIs were obtained before and 90 min after the application of the PCTOC and in vivo LSCIs were obtained for 60 min after the application of the PCTOC. In order to obtain the skin recovery images, saline was applied for 30 min after the application of the PCTOC was completed. The visible appearance of the tubing under ex vivo samples and the in vivo vasculature gradually enhanced over time. The LSCI increased as a function of time after the application of the PCTOC in both ex vivo and in vivo experiments, and properly recovered to initial conditions after the application of saline in the in vivo experiment. The LSCI combined with the PCTOC was greatly enhanced even in deep vasculature. It is expected that similar results will be obtained in in vivo human studies.

• Restorative Dentistry and Endodontics
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• v.30 no.6
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• pp.461-469
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• 2005

The aim of this study was to compare the root canal systems of maxillary and mandibular premolars that had a single root using radiographs and clearing samples. 142 single rooted premolars were selected and mesio-distal and bucco-lingual views were radiographed using intra-oral dental standard films. Four equally trained examiners classify the root canal types from the developed radiographs. After opening the tooth for access, it was stored in $5\%$ NaOCl to dissolve the pulp tissue. Indian ink was then injected into the pulp cavity to stain the pulp tissue. It was cleared in methyl salicylate after being decalcified with $5\%$ nitric acid for 48 hours, and the root canal type was evaluated at a magnification of $\times$ 20 using a stereomicroscope. The results are as follows ; There were statistically significant differences between the radiographs and clearing samples of the root canal types among examiners (p < 0.05). There might be differences in the root canal types among examiners when the same radiograph is used. Therefore, considering the difficulty in estimating the root canal types, clinicians need to be careful when interpreting radiographs before root canal therapy.

• Research in Plant Disease
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• v.26 no.4
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• pp.283-288
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• 2020

In September 2017, vein clearing and yellowing symptoms resembling those caused by viruses were observed on leaves of Malva verticillata in Chungnam, Korea. Nucleic acids were extracted from leaves of five symptomatic plants and tested by reverse transcription polymerase chain reaction using four virus specific primer pairs including malva vein clearing virus (MVCV). Amplicons of the expected size (600 bp) were obtained from total RNA of all samples using the MVCV-specific primers. To confirm the presence of MVCV in symptomatic plants, the DNA fragments from three samples were purified, and directly sequenced. BLAST analysis revealed that it shared the highest nucleotide identity (99%) with a MVCV isolate from tomato (Mexico). The virus isolates obtained from the third re-inoculated Chenopodium was designated as Cm1-5. Tissue from Cm1, Cm3, and Cm5 isolates was mechanically sap inoculated into 23 indicator plants. Cm3 isolate induced chlorotic local and mosaic symptoms in Althaea rosea. Phylogenetic analysis based on coat protein gene of 19 MVCV isolates from 6 different countries and plant species, did not correlated with either the geographical origin of the isolates, or pathogenicity. To our knowledge, this study first reports the natural occurrence of MVCV on M. verticillata in Korea and characterization of three Korean isolates of MVCV.

• Journal of Korean Neurosurgical Society
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• v.37 no.4
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• pp.287-292
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• 2005

Objective: Recent clinical studies have demonstrated that intracisternal administration of recombinant tissue plasminogen activator(rt-PA) can facilitate the normal clearing of blood from the subarachnoid space. Urokinase, a first generation fibrinolytic agent, has been used to liquify such clots with some success. Therefore, recombinant tissue plasminogen activator, a second generation fibrinolytic drug that may be safer and more effective, is studied to evaluate its dosage to lyse clots in vitro and reactivity in the brain parenchyme. Methods: Intracerebral hematomas were created by stereotactically injecting 2ml of clotted autogenous blood into the brain parenchyme of total 28 anesthetized adult cats (weighting 3.8 to 4.1 kg). The control animals (group A) received 1 ml of normal saline injected into the clots and the experimental animals received each 0.1 mg of rt-PA (group B), 0.5mg of rt-PA (group C) and 1 mg of rt-PA (group D) at 6 hours after the clot injection. Results: 1. The amount of remained clots after lysing the hematomas were as follows: $1.80{\pm}0.17ml$ in group A, $1.65{\pm}0.23ml$ in group B, $0.61{\pm}0.37ml$ in group C and $0.52{\pm}0.34$ in group D. The result indicated that hematomas in rt-PA treated groups (C & D) were lysed better than the control group. 2. At least 0.5mg of rt-PA should be required for the lysis of 2ml of hematomas. 3. Light microscopic examination revealed no histological evidence of hemorrhage in tissue sections from each brain. Conclusion: Recombinant tissue plasminogen activator may be safely and effectively employed for the lysis of intracerebral hematomas in animal model.