• Applied Microscopy
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• 제23권2호
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• pp.11-26
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• 1993

This experiment was performed to study the morphological responses of the pigment epithelial cell and the Bruch's membrane of the retina of rat following X-ray irradiation. Male rats were divided into normal and experimental groups. The heads of the rats, under sodium thiopental anesthesia, were exposed to 3,000 rads or 6,000 rads of radiation in a single dose, respectively. The source was a Mitsubishi Linear Accelerator ML-4MV. The target to skin distance was 80cm, and the. dose rate was 200 rads/min. The experimental groups were sacrificed on the 6th hour, 2nd and 6th day after X-ray irradiation. Under anesthesia, 1% glutaraldehyde-1% paraformaldehyde solution(0.1M Millonig's phosphate buffer, pH 7.3) was perfused through the left ventricle and ascending aorta. Pieces of the tissue taken from the posterior region of the retina were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde(0.1M Millonig's phosphate buffer, pH 7.3) and 1% osmium tetroxide(0.1M Millonig's phosphate buffer, pH7.3), and embedded in araldite mixture. The ultrathin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100 CX-II electron microscope. The results were as follow; 1. The morphological changes of the pigment epithelial cells were not pronounced after exposure to 3,000 rads of X-ray. But on the 6th hour after exposure to 6,000 rads of X-ray, bulging nuclear membrane protruding into the cytoplasm and nuclear chromatin clumped into numerous masses along the nuclear membrane were observed. At the 2nd and 6th day post-irradiation, partial cytolysis or necrosis were seen. 2. The thickness of the Bruch's membrane of the experimental groups were increased in the time and dose range covered by this study, and splitting or diffusing basal laminae of the choriocapillary layer were observed frequently in the experimental group. Above results suggest that large amount(6,000 rads) of head irradiation induce direct hazardous effects on the pigment epitherial cells and Bruch's membrane of the retina of the rat, but pigment epithelial cells are more radioresistant than Bruch's membrane.

• Journal of Periodontal and Implant Science
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• 제23권1호
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• pp.77-96
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• 1993

정상치아와 이환치아 및 구연산에 의하여 탈회한 치근면에대한 치주인대세포의 부착상태를 비교관찰 하기 위하여 정상치아와 이환치아를 취하여 정상군, 이환치근의 치근면활택술군 및 구연산처리군으로 분류하고 시험관적 실험을 통하여 관찰하였다. 세포부착실험전 각 군의 치근면의 형태를 주사전자현미경을 이용하여 관찰하였으며, 사람의 정상소구치에서부터 얻어진 치주인대세포를 배야하여 각 군의 절편을 함유한 조직배양기에 ml당 $4.5{\times}\;10^4$개의 세포를 가진 배양액 1ml씩을 넣고 동일조건하에서 30분, 1시간, 2시간, 6시간, 12시간 및 24시간 동안 배양한 후 세포의 부착이 일어난 후 상태를 주사전자현미경을 이용하여 관찰하였고, 세포증식정도를 알아보기 위하여 각 절편에 $5{\times}10^4$개의 세포가 함유된 배양액 1ml씩을 넣고 6시간동안 배양 후, 새 배양기에 옮기고 24, 48, 72시간동안 배양하여 trypsin 처리로 세포를 분리시킨 후 광학위상차 현미경을 이용, 치근단위면적당 증식된 세포수를 측정하여 다음과 같은 결과를 얻었다. 세포부착실험전 각 군의 치근면 형태를 관찰하였을 때 구연산처리군에서는 교원섬유의 노출부위라고 여겨지는 미세한 돌출구조들을 관찰할 수 있었으며 상아세관의 노출부위라고 여겨지는 함몰양상이 치근활택술군에 비하여 현저하였고 정상치아군에서는 이러한 양상을 관찰할 수 없었다. 실험 각 군 및 대조군 사이에서 세포들의 형태적인 차이점은 분명하지 않았으나 구연산처리군에서 다른 군들보다 초기의 세포부착양상이 다소 빠르게 진행 됨이 관찰되었다. 세포배양 개시 후 6시간이 경과 한 후에는 모든 군에서 공히 세포의 형태가 초기의 구형의 형태에서 편평한 세포의 형태로 변화되기 시작함이 관찰 되었고 24시간 이후에는 모든 군에서 세포들이 치근면에 납작하게 부착되고 일반적인 섬 유아세포의 형태로 변화되어 셰포의 전개가 완성된 양상이 보였다. 세포증식율의 실험에서는 24시간 후 증식된 세포 수는 치근면활택술군에서 가장높게 나타났으며 정상군에서 가장 낮은 수치를 보였으며 48시간 및 72시간후 측정에서는 구연산처리군이 다른 군들에 비해 더 많은 수의 세포증식을 관찰할 수 있었으며 각 군간의 차이는 통계학적으로 유의하였다.

• Journal of Periodontal and Implant Science
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• 제25권3호
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• pp.587-602
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• 1995

치근면처리후 신부착에 관여하는 치주인대 세포의 부착을 비교하기위해 치근활택술만 시행한 군, 치근활택술후 구연산처리한 군, 치근활택술후 테트라사이클린처리한 군으로 분류하여 치주인대세포에 대한 부착양상을 관찰하였다. 세포증식실험에서는 각군의 시편을 조직배양지에 넣고 $1{\times}10^5$ cells/ml 농도의 치주인대세포를 1 ml씩 넣어 6시간 배양후 새 배양기에 옮겨 24시간, 28시간 및 72시간동안 배양하여 세포수를 측정하였고 세포부착후 주사전자현미경 관찰에서는 각각의 조직배양기에 1${\times}$104 cells/ml의 치주인대세포를 가진 배양액 1 ml씩을 넣어 배양하여 세포배양개시 푸 30분, 6시간, 24시간에 고정한후 탈수하고 임계점으로 건조시켜 주사전자현미경을 사용하여 세포부착양상을 관찰하였다. 세포증식실험에서 부착된 세포수가 24, 48, 72시간 모두에서 치근활택군, 구연산처리군, 테트라사이클린처리군순으로 높았고 치근활택군과 테트라사이클린처리군사이에 통계학적 유의성이 있었으며 24, 48시간에서는 구연산처리군과 테트라사이클린처리군사이에 통계학적 유의성이 있어 탈회군이 비탈회군보다 증가된 세포증식을 보였고 테트라사이클린처리군이 구연산처리군보다 증식된 세포수가 많았다. 주사전자현미경관찰에서 모든 군이 24시간 경과 후 전개가 거의 완성되는 양상을 보였고 테트라사이클린처리군의 초기전개가 다소 빠르게 진행됨이 관찰되었으며 테트라사이클린처리군과 구연산처리군이 세포가 부착되는 양상에서 치근활택군보다는 전개가 다소 진전된 양상을 나타내었으나 두 군간에는 유사한 전개가 진행되어 탈회면과 비탈회면의 차이는 나타나나 두 군간의 뚜렷한 차이는 보이지 않았다. 이상의 연구결과 테트라사이클린과 구연산처리가 치근활택술만 시행한 경우보다 치주인대세포의 증식 및 전개에 더 유리하며, 테트라사이클린과 구연산은 치주인대세포의 전개양상은 유사하나 치주인대세포증식에 있어서는 테트라사이클린에서 더 나은 효과가 있었다.

• Applied Microscopy
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• 제24권4호
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• pp.61-77
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• 1994

The calcium-binding proteins (CaBP), parvalbumin (PV) and calbindin-D 28K (calbindin) are particularly abundant and specific in their distribution, and present in different subsets of neurons in many brain regions. Although their physiological roles in the neurons have not been elucidated, they are valuable markers of neuronal subpopulations for anatomical and developmental studies. This study is designed to characterize dorsal lateral geniculate nucleus (dLGN) neurons and axon terminals in terms of differential expression of immunoreactivity (IR) for two well-known CaBPs, PV and calbindin. The experiments were carried out on 6 adult monkeys. Monkeys were perfused under deep Nembutal anesthesia with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer. After removal, the brains were postfixed for 6-8 hr in 2% paraformaldehyde at $4^{\circ}C$ and infiltrated with 30% sucrose at $4^{\circ}C$. Thereafter, they were frozen in dry ice. Serial sections of the thalamus, at $20{\mu}m$, were made in the frontal plane with a sliding microtome. The sections were stained for PV and calbindin with indirect immunocytochemical methods. For electron microscopy, after infiltration with 30% sucrose the blocks of thalamus were serially sectioned at $50{\mu}m$ with a Vibratome in the coronal plane and stained immediately by indirect ABC methods without Triton X-100 in incubation medium. Stained sections were postfixed in 0.2% osmium tetroxide, dehydrated and flat-embedded in Spurr resin. The block was then trimmed to contain only a selected lamina or interlaminar space. The dLGN proper showed strong PV IR in fibers in all laminae and interlaminar zones. Particularly dense staining was noted in layers 1 and 2 that contain many stained fibers from optic tract. Neuronal cell body stained with PV was concentrated only in the laminae. In these laminae staining was moderate in cell bodies of all large and medium-sized neurons, and was strong in cell bodies of some small neurons together with their processes. Calbindin IR was marked in the neuronal cell body and neuropil in the S layers and interlaminar zones whereas moderate in the neuropil throughout the nucleus. Regional difference in distribution of PV and calbindin IR cell is distinct; the former is only in the laminae and the latter in both the S layer and interlaminar space. The CaBP-IR elements were confined to about $10{\mu}m$ in depth of Vibratome section. The IR product for CaBP was mainly associated with synaptic vesicle, pre- and post-synaptic membrane, and outer mitochondrial membrane and along microtubule. PV-IR was noted in various neuronal elements such as neuronal soma, dendrite, RLP, F, PSD and some myelinated or unmyelinated axons, and was not seen in the RSD and glial cells. Only a few neuronal components in dLGN was IR for calbindin and its reaction product was less dense than that of PV, and scattered throughout cytoplasm of soma of some relay neurons, and was also persent in some dendrite, myelinated axons and RLP. The RSD, F, PSD and glial elements were always non-IR for calbindin. Calbindin labelled RLP were presynaptic to unlabeled dendrite or dendritic spine and PSD. Calbindin-labeled dendrite of various sizes were always postsynaptic to unlabeled RSD, RLP or F. From this study it is suggested that dLGN cells of different functional systems and their differential projection to the visual cortex can be distinguished by differential expression of PV and calbindin.

• Applied Microscopy
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• 제24권2호
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• pp.78-92
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• 1994

Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.

• Applied Microscopy
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• 제35권3호
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• pp.153-163
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• 2005

이 연구는 생쥐의 샅부위에 Ehrlich 암세포를 이식한 후 BCG (Bacillus Calmette-Guerin)를 투여하였을 때, 위점막 으뜸세포의 미세구조에 미치는 영향을 알아보기 위하여 시행하였다. 실험동물로는 체중 25 g 내외의 ICR생쥐를 사용하였으며 정상대조군과 실험군으로 구분하였다. 정상대조군 이외의 실험군 동물들은 샅부위 피부밑조직에 각각 $1x10^7$개의 Ehrlich암세포를 이식한 후, 다음날부터 BCG 투여군은 BCG를 피부밑조직에 격일 간격으로 7회 투여했으며, 종양대조군은 암세포이식 후에 약제 대신 0.2 mL의 생리식염수를 피부밑조직에 격일간격으로 7회 주사하였고, 정상대조군은 암세포를 이식하지 않은 동물을 사용하였다. 모든 실험군은 마지막 주사한 다음날 오전 10시에서 11시 사이에 ether마취하에 앞배벽을 열어 위조직을 절취하였다. 절취한 조직은 통상적인 방법으로 전자현미경관찰을 위한 표본을 작성한 후, JEM 100 CX-II 전자현미경으로 비교 관찰하였다. 각 세포는 5,300 배율로 촬영한 후 3배로 확대 인화한 사진을 비교 관찰하였으며, 각 실험군마다 200개 이상의 과립의 지름과 사립체의 크기(장경)를 측정하였으며 student-t test를 이용하여 통계처리 하였다. BCG 투여군의 으뜸세포는 과립세포질세망과 골지복합체의 수조가 다소 팽대되고 수초구조가 다소 많이 관찰되는 이외는 정상대조군에 비해 큰 차이가 없었다. 으뜸 세포의 분비과립의 크기는 정상대조군, 종양대조군 및 BCG 투여군의 경우 각각 $0.98({\pm}0.108){\mu}m,\;1.05({\pm}0.092){\mu}m$ 및 $0.93({\pm}0.053){\mu}m$ 였다. 으뜸세포의 사립체의 크기는 정상대조군, 종양대조군 및 BCG 투여군이 각각 $0.80({\pm}0.130){\mu}m,\;0.83({\pm}0.143){\mu}m$ 및 $0.72({\pm}0.078){\mu}m$ 였다. 이상의 결과를 종합해보면 BCG를 반복 투여하면 위점막으뜸세포의 분비과립이 약간 작아지는 등 분비기능이 다소 억제되나 그 정도가 경미하여 으뜸세포의 분비기능에 큰 손상을 주지 않는 것으로 생각된다.

• Applied Microscopy
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• 제7권1호
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• pp.21-43
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• 1977

This experiment was undertaken in order to localize the labeled dbcAMP (dibutyryl cyclic AMP) in oocytes whose development has been suppressed by cold dbcAMP for 6 or 19 hours in vitro. Mouse oocytes were obtained from the ovaries of 3-4 week old A strain female mice, by puncturing the Graafian follicles in the modified Krebs-Ringer bicarbonate salt solution under the dissecting microscope. Those oocytes which have intact germinal vesicle were cultured in the basic culture medium supplemented with 0.4% bovine serum albumin (BSA). Cultivation of the oocytes was carried out in a microtube developed by Cho (1974). The cultures were then incubated in a humidified 5% $CO_2$ incubator maintained at $37^{\circ}C$ for 6 or 19 hours (Donahue, 1968). DbcAMP was added to culture medium for a final concentration of 100ug/ml, and $^3H-dbc$ AMP (specific activity 13 Ci/mM) for a final concentration of $40{\mu}Ci/ml$ was also added to the medium. For electron microscopic autoradiography, those oocytes recovered from the culture were washed with phosphate buffer (pH 7.4), and immediately prefixed in a 2.5% glutaraldehyde overnight and postfixed for 2 hours at $4 ^{\circ}C$ in 1% osmium tetroxide in phosphate buffer with pH 7.4 (Palade, 1952). After fixation, the materials were dehydrated in graded alcohol series and embedded in Epon 812 mixture based on the standard procedures (Luft, 1961). The thin sections $600-700{\AA}$ thick were mounted on the grids of 200 meshes. The grids containing sections were coated with a nuclear emulsion Kodak NTB-3 and stored in a cold dark box (at $4^{\circ}C$) for 3 weeks. After exposure, the samples were developed with Kodak D-19 and stained with uranyl acetate and lead citrate. Routine observation was made with Hitachi HU-11E electron microsocope. The results of the observation were as followings: 1. It was found that the labeled dbcAMP penetrated the egg plasma membrane and dispersed at random in the cytoplasm. 2. It was also observed that most of the labeled dbcAMP was attached to microfibrillar lattices portion of the oocyte cytoplasm. There fore, it is presumed that the receptor of the dbcAMP is localized in the microfibrillar lattices of the oocyte. 3. It also seems that some other cell organells such as mitochondria, Golgi complex, cortical granules are not directly related to the action of the dbcAMP. 4. The labeled dbcAMP was neither observed in the membrane nor in the nucleus. Therefore, it seems that there is no relationship between the concentration of dbcAMP and the nuclear membranous permeability. 5. There was no difference in number of dbcAMP particles when oocytes were cultured for 6 hours and 19 hours. 6. However, it was observed that, in same of the oocytes suppressed in germinal vesicle by dbcAMP for 19 hours, cell organells were moved and concentrated to a small portion of the cytoplasm, and that the morphology of the organells greatly changed to an abnormal. form. Therefore, it is supposed that those oocytes were in the process of degeneration. From the above results, it is expected that dbcAMP penetrated the egg membrane and was bound to the receptor which seems to be located in the microfibrillar lattiees portion, and that this dbcAMP-receptor complex inhibited some enzyme system of the oocytes which are essential for the germinal vesicle breakdown.

• Applied Microscopy
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• 제41권1호
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• pp.1-11
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• 2011

이 실험은 Ehrlich 종양세포를 이식한 후 5-fluorouracil, mitomycin 및 acriflavine-guanosin 복합제(AG60)을 투여하였을 때, 위점막 점액상피세포의 미세구조적 변화를 연구하고자 시행하였다. 실험동물로는 ICR생쥐를 사용하였으며 정상대조군 이외의 종양대조군, 5-fluorouracil, mitomycin C 및 AG60 투여군의 동물들은 샅부위 피부밑조직에 각각 $1{\times}10^7$의 Ehrlich 종양세포를 이식하였다. 각각의 실험군은 종양세포를 이식한 다음날부터 5-fluorouracil (30 mg/kg), mitomycin C ($400{\mu}g/kg$) 및 AG60 (30 mg/kg)을 격일 간격으로 한번씩 피부밑조직에 주사하였다. 종양대조군은 종양세포이식 후에 0.2 mL의 생리식염수를 피부밑조직에 주사하였고 정상대조군은 종양세포를 이식하지 않은 동물을 사용하였다. 종양대조군을 비롯한 실험군은 생리식염수 또는 각각의 약제를 격일 간격으로 7회씩 투여한 다음날, 에테르(ether) 마취하에 앞배벽을 열어 위조직을 절취하였다. 절취한 조직은 2.5% 글루타르알데히드(glutaraldehyde)-1.5% 파라포름알데히드(paraformaldehyde) 혼합액에 고정한 후, 1% 오스뮴사산화물(osmium tetroxide)용액에 다시 고정한 후 탈수과정을 거쳐 애럴다이트(araldite) 혼합액에 포매하였다. 포매된 위점막 조직은 얇은 절편을 만들었으며, 각 절편은 우라닐아세테이트(uranyl acetate)용액과 구연산납(lead citrate)용액으로 대조염색한 후, 전자현미경으로 비교 관찰하였다. 5-fluorouracil 투여군은 종양대조군에 비해 점액상피세포에 수초구조(myelin figure)가 자주 관찰되고, 분비과립이 세포질을 포함한 막성구조에 싸여 속공간으로 함께 분비되는 부분분비현상이 보일 정도로 손상을 받았다. 그리고 mitomycin C 역시 수초구조가 자주 관찰되고 분비과립을 함유한 세포질이 속공간으로 돌출되는 등의 미세구조적 변화를 보였다. 그러나 AG60의 경우는 수초구조와 다소포체가 비교적 자주 관찰되는 외에는 별다른 미세구조적 변화를 관찰할 수 없었다. 이상의 결과를 종합해보면 mitomycin C, 5-fluorouracil 및 AG60을 반복 투여 하면 위점막 점액상피세포의 분비기능이 억제되었음을 시사하는 미세구조적 변화를 보였다. 그러나 세포의 손상 정도가 mitomycin C와 5-fluorouracil 투여군에 비해 AG60 투여군에서 매우 경미하였으므로 AG60은 위점막 점액상피세포의 분비기능에 큰 손상을 주지 않는 약제라고 생각된다.

• Applied Microscopy
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• 제37권3호
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• pp.157-166
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• 2007

Ehrlich 암세포를 샅부위에 이식한 후 BCG를 투여하였을 때 생쥐 막창자꼬리점막 흡수세포의 미세구조적 변화를 관찰하였다. 실험동물로는 체중 25 gm내외의 성숙한 생쥐 (ICR계통)를 정상대조군, 암세포이식대조군, BCG투여군으로 구분하였다. 암세포이식대조군과 BCG투여군 동물들은 샅부위 피부밑조직에 각각$1{\times}10^7$의 Ehrlich 암세포를 주사하여 이식하였다. 암세포이식대조군은 암세포이식 후에 약제 대신 0.5mL의 생리식염수를, BCG 투여군은 일정량(0.5 mL/25 gm B.W.: $0.03{\times}10^8{\sim}0.32{\times}10^8CFU$)의 BCG를 하루건너 한 번씩 7번 피부밑조직에 주사하였다. 절취된 막창자꼬리조직은 통상적인 방법으로 고정 및 탈수과정을 거쳐 araldite 혼합액에 포매하여 절편을 만든 다음 JEM 100CX-II(JEOL, Japan)전자현미경으로 비교 관찰하였다. 정상군 막창자꼬리점막의 흡수세포는 미세융모, 골지복합체, 과립세포질세망 및 자유면쪽의 미세소포 등, 미세구조가 전형적인 소화관 흡수세포의 것과 유사하였다. 암세포이식대조군의 흡수세포는 자유면쪽에 미세융모가 매우 조밀하게 형성되고 작은 소포들이 다수 관찰되고, 다수의 사립체, 잘 발달된 골지복합체, 과립세포질세망 및 용해소체를 포함하고 있는 모습이 정상대조군의 모습과 유사하였다. BCG투여군의 흡수세포는 세포소기관의 미세구조를 비롯한 전체적인 모습이 정상대조군의 모습과 유사하였으나 세포질 내에 말이집구조를 포함하고 있는 세포들이 자주 관찰되었으며, 사립체에 전자밀도가 높은 과립모양의 구조를 포함하고 있는 세포들이 비교적 자주 관찰되었다. 이상의 결과를 종합하여 보면 BCG를 반복 투여하면 막창자꼬리점막의 흡수세포는 미세구조에 다소 손상을 받아 흡수기능이 다소 억제되나 그 정도가 매우 경미하였으므로 BCG는 막창자꼬리점막 흡수세포의 기능에 큰 손상을 주지 않는다고 생각된다.

• Restorative Dentistry and Endodontics
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• 제23권1호
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• pp.141-161
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• 1998

The depth and patterns of demineralization according to the difference in concentration and application time of phosphoric acid were observed through the transmission electron microscope, and shear bond strengths to the acid -conditioned dentin were then measured and compared with the TEM results. To investigate the influence of polymer addition into the phosphoric acid and the effect of difference in concentration and application time of the acid, the specimens were randomly divided into 9 groups. Among the specimens, the exposed dentin surfaces were acid-conditioned with 10% polymer-thickened phosphoric acid(All Bond 2, Bisco, U.S.A.) and aqueous 10%, 20%, 30%, 40% phosphoric acid for 20 seconds, The rest of the specimens were acid-conditioned with 10% phosphoric acid for 15s, 30s, 60s, 120s respectively. The specimens were immersed in 4% glutaraldehyde in 0.1M sodium cacodylate buffer and postfixed with 1 % osmium tetroxide without decalcification and then observed under a JEOL Transmission Electron Microscope(JEM 1200 EX II, Japan). After the specimens were acid-conditioned as the above, primer and adhesive resin were applied to blot-dried dentin and shear bond strengths were then measured and analysed. The results were as follows : 1. The intertubular demineralization depth of 4.0-$5.0{\mu}m$ in 10% polymer-thickened phosphoric acid gels was similar or slightly deeper than that of 4.0-$4.5{\mu}m$ in aqueous 10% phosphoric acid solution. 2. The intertubular demineralization depth of aqueous 20%, 30% and 40% phosphoric acid solution was 6.5-$7.0{\mu}m$, 6.5-$7.5{\mu}m$ and 9.0-$15.0{\mu}m$ respectively. It showed that the depth of dentin demineralization is partly related to the concentration of phosphoric acid solution. 3. The intertubular demineralization depth of aqueous 10% phosphoric acid solution in application time for 15s, 30s, 60s and 120s was 2.5-$3.0{\mu}m$, 4.0-$6.0{\mu}m$, 6.5-$7.0{\mu}m$ and 8.5-$14.0{\mu}m$ respectively. It showed that the depth of dentin demineralization is directly related to the application time of phosphoric acid solution. 4. The partially demineralized dentin layer between demineralized collagen layer and unaffected dentin was showed to a width of 0.5-$1.0{\mu}m$ in lower concentration groups treated with aqueous 10% phosphoric acid for 20s, 60s, 120s and 20% phosphoric acid for 20s. 5. The demineralization effect at the border of intertubular-peritubular junction was less evident than that in the peritubular and intertubular dentin. The collagen fibers in the intertubular dentin had a random orientation, whereas those that lined the tubules were circumferentially aligned. The cross-linkage of dentinal collagen in demineralized collagen layer was clearly seen. 6. A statistically significant difference of bond strengths according to the difference in phosphoric acid concentration did not exist among the groups treated with 10%, 20%, 30% and 40% acid solution (P>0.05). However, bond strengths to the treated dentin with 10% phosphoric acid solution for 30s were significantly higher than that for 120s (P<0.05).