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

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.46 no.7
• /
• pp.857-867
• /
• 2017

This study was performed to encapsulate low molecular weight marine collagen and ${\gamma}$-aminobutyric acid (GABA)-producing lactic acid bacteria to inhibit degradation and improve survival rate during exposure to adverse conditions of the gastro-intestinal tract. Calcium-alginate method was used for the manufacture of a double-layered coated capsule. The inner core material was composed of collagen and lactic acid bacteria, and the coating materials were alginate and chitosan. The sizes and shapes of the double-coated capsule were affected mainly by centrifuge speed and pH. Manufactured capsules were observed with a scanning electron microscope and by confocal laser scanning microscopy to confirm the micromorphological changes of capsules and bacterial cells. As a result, double-layered coated capsules were not degraded at pH 1.2, whereas degradation occurred at pH 7.4. In addition, GABA and collagen were maintained in stable state at pH 1.2. Therefore, double-layered coated capsules developed in this study would not be degraded in the stomach and could be stably delivered to the small intestine to benefit intestinal and dermatic health.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
• /
• v.29 no.1
• /
• pp.105-117
• /
• 1999

Purpose: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca/sup 2+/] can lead to DNA fragmentation by Ca/sup 2+/-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca/sup 2+/] was measured only by minutes or hours respectively. We need to evaluate [Ca/sup 2+/] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells. Materials and Methods: We have measured [Ca/sup 2+/] in human gingival epitheloid cancer cell with 10Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca/sup 2+/ indicator dye, fura-2. In order to find out that the transient rise in [Ca/sup 2+/] could induced apoptosis, cells were incubated for 1 hour at 37℃ with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells. Results: After irradiation, the transient and temporal increasing of [Ca/sup 2+/] in the KB cells was founded. Though, there was no change in the intracellular [Ca/sup 2+/] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group. Conclusion: In KB cells there were incipient and temporal increasing of the [Ca/sup 2+/] with 10Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.

• Journal of the Korean Chemical Society
• /
• v.54 no.4
• /
• pp.451-459
• /
• 2010

The fluorescence detection of intracellular metal ions are high interest in the fields of organic molecular chemistry and cellular biology. This study was purposed to detection for mercury and zinc in the cell using fluorescent chemosensor (FS). FS exhibits a weak fluorescence, but emits strong fluorescence upon Zn$^{2+}$ complexation. The increased fluorescence of the 2FS/Zn$^{2+}$ can be quenched completely by addition of only 1 equiv of Hg$^{2+}$ with the formation of complex FS-Hg$^{2+}$. Four cell lines (LLC-MK2, Hela, HT29 and AMC-HN3) were used for fluorescence imaging by confocal microscope. The cell viability MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was evaluated after cell treatment of FS, Zn$^{2+}$, FS-Zn$^{2+}$, Hg$^{2+}$ on LLC-MK2 cell line. The cytotoxicity of FS was showed to viability over 80%. This study has shown that FS can be detected for selective imaging of Zn$^{2+}$ and Hg$^{2+}$ in living cells.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.33 no.2
• /
• pp.93-97
• /
• 2007

Even in the moderately concentrated anionic surfactant system, some special encapsulation method can shield the papain enzyme from proteolytic attacks. The stabilization of enzyme has been a major issue for successful therapies. In this study, we first stabilized an enzyme, papain in the microcapsules by using polyols, polyethyleneglycol (PEG), poly-propyleneglycol (PPG), and PEG-PPG-PEG block copolymer. In the analysis of EDS and CLSM, it was demonstrated that polyols are effectively located in the interface of papain and polymer. Polyols located in the interface had an ability to buffer the external triggers by hydrophobic partitioning, preventing consequently the catalytic activity of papain in the micro-capsules. Second. we introduced multi-layer capsulation methods containing ion complex. Such a moderately concentrated anionic surfactant system as wash-off cleansers, surfactants and waters can cause instability of entrapped enzymes. Surfactants and water in our final products swell the surface of enzyme capsules and penetrate into the core so easily that we can not achieve the effect of enzyme, papain. In this case, the ion complex multi-layer capsule composed of sodium lauroyl sarcosinate and polyquaternium-6 could effectively prevent water from penetration into the core enzyme, followed by in vivo test, and evaluate the stratum corneum (SC) turn-over speed.

• Archives of Pharmacal Research
• /
• v.29 no.10
• /
• pp.859-865
• /
• 2006

With an approach to study the anti-tumor effects and mechanism of selenium compound, we investigated the anti-tumor activity and mechanism of $Na_5SeV_5O_{18}{\cdot}3H_2O$ (NaSeVO) in K562 cells. The results showed that $0.625{\sim}20\;mg/L$ NaSeVO could significantly inhibit the proliferation of K562 cells in vitro in a time- and concentration-dependent manner as determined by microculture tetrazolium (MTT) assay, the IC50 values were 14.41 (4.45-46.60) and 3.45 (2.29-5.22) mg/L after 48 hand 72 h treatment with NaSeVO respectively. In vivo experiments demonstrated that i.p. administration of 5, 10 mg/kg NaSeVO exhibited an significant inhibitory effect on the growth of transplantation tumor sarcoma 180 (S180) and hepatoma 22 (H22) in mice, with inhibition rate 26.8% and 58.4% on S180 and 31.3% and 47.4% on H22, respectively. Cell cycle studies indicated that the proportion of G0/G1 phase was increased at 2.5 mg/L while decreased at 10 mg/L after treatment for 24, 48 h. Whereas S phase was decreased at 2.5-5 mg/L and markedly increased at 10 mg/L after treatment for 48 h. After treatment for 24 h, 10 mg/L NaSeVO also markedly increased S and G2/M phases. Take together, the result clearly showed that NaSeVO markedly increased S and G2/M phases at 10 mg/L. The study of immunocytochemistry showed that the expression bcl-2 is significantly inhibited by 10 mg/L NaSeVO, and bax increased. Morphology observation also revealed typical apoptotic features. NaSeVO also significantly caused the accumulation of $Ca^{2+}$ and $Mg^{2+}$, reactive oxygen species (ROS) and the reduction of pH value and mitochondrial membrane potential in K562 cells as compared with control by confocal laser scanning microscope. These results suggest that NaSeVO has anti-tumor effects and its mechanism is attributed partially to apoptosis induced by the elevation of intracellular $Ca^{2+}$, $Mg^{2+}$ and ROS concentration, and a reduction of pH value and mitochondria membrane potential (MMP).

• The Journal of Korean Academy of Prosthodontics
• /
• v.39 no.1
• /
• pp.71-83
• /
• 2001

The purpose of this study was to compare the effects of various surface treatments by measuring removal torque on bone healing around titanium implants. 40 Screw-shaped cp titanium implants with length of 4mm, outer diameter of 3.75mm, and pitch-height of 0.5mm were used Group 1 was left as machined(control), Group 2 was blasted with $50{\mu}m\;Al_2O_3$, group 3 was blasted and etched in etching solution($NH_4OH : H_2O_2:H_2O= 1 : 1 : 5$) at $90^{\circ}C$ for 1 minute group 4 was blasted and oxidated under pure oxygen at $800^{\circ}C$. The implant surface roughness was analyzed with SEM and CLSM(Confocal Laser Scanning Microscope) and implants were placed in proximal tibial metaphysis of 10 New Zealand White rabbits. After 3 months of healing period, removal torque of each implant was measured to compare bone healing around implant. The results obtained were as follows 1. In SEM view, blasting increased the roughness of the surface, but etching of that rough surface decreased the roughness due to the removal of the tip of the peak. Oxidation also decreased the roughness due to formation of needle-like oxide grains on the implant surface. 2. The Sa value from CLSM was least in the machined group($0.47{\mu}m$), greatest in blasted group($1.25{\mu}m$), and the value decreased after etching($0.91{\mu}m$) and oxidation($0.94{\mu}m$). 3. The removal torque of etched group(24.5Ncm) was greater than that of machined group(16.7Ncm) (P<0.05), and was greatest in the oxidated group(40.3Ncm) and the blasted group(34.7Ncm).

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.3
• /
• pp.301-309
• /
• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.

• Korean Journal of Microbiology
• /
• v.50 no.4
• /
• pp.360-367
• /
• 2014

Candida albicans is an opportunistic and the most prevalent fungal pathogen that can cause superficial and systemic infections in immunocompromised patients. C. albicans can promote the transition from budding yeast to filamentous form, generating biofilms. Infections associated with C. albicans biofilms are frequently resistant to conventional antifungal therapy. Therefore, the development of more effective antifungal drugs related with biofilm formation is required urgently. The roots of Rheum undulatum have been used for medicinal purposes in Korea and China traditionally. The aim of present study was to evaluate the effect of R. undulatum extract upon preformed biofilms of 12 clinical C. albicans isolates and the antifungal activities. Its effect on preformed biofilms was evaluated using XTT reduction assay, and metabolic activity of all tested strains was reduced significantly ($49.4{\pm}6.0%$) at 0.098 mg/ml R. undulatum. The R. undulatum extract blocked the adhesion of C. albicans biofilms to polystyrene surfaces, and damaged the cell membrane integrity of C. albicans which was analyzed by CFDA, AM, and propidium iodide double staining. It caused cell lysis which was observed by Confocal laser scanning and phase contrast microscope after propidium iodide and neutral red staining, respectively. Membrane permeability was changed as evidenced by crystal violet uptake. The data suggest that R. undulatum inhibits biofilm formation by C. albicans, which can be associated with the damage of the cell membrane integrity, the changes in the membrane permeability and the cell lysis of C. albicans.

• Restorative Dentistry and Endodontics
• /
• v.25 no.2
• /
• pp.243-253
• /
• 2000

The purpose of this in vitro study was to evaluate dentin bonding by two different dentin bonding systems(DBS) using acetone based primer or adhesive [All Bond 2(AB2), One Step(OS)] when they were applied by wet or dry bonding technique. Morphology of resin-dentin interface and hybrid layer thickness(HLT) were investigated using Confocal Laser Scanning Microscope(CLSM) and compared to shear bond strength(SBS). 72 extracted sound human molars were randomly divided into 4 groups of 18 teeth each - Group 1.(AW); AB2 by wet bonding. Group 2(AD); AB2 by dry bonding. Group 3.(OW); OS by wet bonding, Group 4.(OD); OS by dry bonding. In 6 teeth of each group, notch-shaped class V cavities(depth 2mm) were prepared on buccal and lingual surface at the cementoenamel juction(12 cavities per group). To obtain color contrast in CLSM observation, bonding resins of each DBS were mixed with rhodamine B and primer of AB2 was mixed with sodium fluorescein. Prepared teeth of each group were treated with AB2, OS, respectively according to the manufacturer's instructions except for dentin surface moisture treatment after acid etching. In group 1 and 3, after acid etching, excess water was removed with wet tissue(Kimwipes), leaving consistently shiny, visibly hydrated dentin surface. In group 2 and 4, dentin surface was dried for 10 seconds at 1 inch distance. The treated teeth were then packed with composite resin(${\AE}$litefil) and light-cured. 12 microscopic samples($60{\sim}80{\mu}m$ thickness) of each group were obtained after longitudinal section and grinding(Exakt cutting and grinding system). Morphological investigation of resin-dentin interface and HLT measurement using CLSM were done. For measurement of SBS, remaining 12 teeth of each group were flattened occlusally to remove all enamel and grinded to 500 grit SiC(Pedemet Specimen Preparation Equipment). After applying DBS on the exposed dentin surface, composite resin was applied in the shape of cylinder, which has 5mm diameter, 1.5mm thickness, and light cured. SBS was measured using Instron with a crosshead speed of 0.5mm/min. It was concluded as follows, 1. HLT of AW(mean: $2.59{\mu}m$) was thicker than any other group, and followed by AD, OW, OD in descending order(mean; 2.37, 2.28, $1.92{\mu}m$). Only OD had statistically significant differences(p<0.05) to AW and AD. 2. There were intimate contact of resin and dentin at the interface in wet bonding groups, but gaps or irregular interfaces were observed in dry bonding groups. 3. The length, diameter, density of resin tags were various even in the same group without significant differences between groups and lots of adhesive lateral branches were observed. 4. There were no statistically significant difference of SBS between AB2 and OS, but SBS of wet bonding groups were significantly higher(p<0.05) than dry bonding groups. 5. There were no consistent relationships between HLT and SBS.