激光扫描共焦显微镜活体组织诊断技术的应用进展

激光扫描共焦显微镜作为一种新的无创诊断技术,能对人体体表组织细胞在活体进行观察,相对于常规组织学检查,它不需要标本切取、固定、包埋、切片、染色等繁琐的过程,具有很大的优势。它能在活体组织观察细胞形态和组织结构,甚至在使用增强对照的试剂后,能在分子水平观察细胞的活动。口腔黏膜、体表皮肤由于其特殊的位置及结构,使激光扫描共焦显微镜在这些方面得到广泛的应用。作者对激光扫描共焦显微镜在口腔黏膜、皮肤病变的活体组织诊断以及外科手术中的应用进行综述。     

Comparison of fungiform taste‐bud distribution among age groups using confocal laser scanning microscopy in vivo in combination with gustatory function

The aim of this study was to compare the distribution of taste buds in fungiform papillae (FP) and gustatory function between young and elderly age groups. Confocal laser scanning microscopy was used because it allows many FP to be observed non‐invasively in a short period of time. The age of participants (n = 211) varied from 20 to 83 yr. The tip and midlateral region of the tongue were observed. Taste buds in an average of 10 FP in each area were counted. A total of 2,350 FP at the tongue tip and 2,592 FP in the midlateral region could be observed. The average number of taste buds was similar among all age groups both at the tongue tip and in the midlateral region. The taste function, measured by electrogustometry, among participants 20–29 yr of age was significantly lower than that in the other age groups; however, there was no difference among any other age groups in taste function. These results indicate that the peripheral gustatory system is well maintained anatomically and functionally in elderly people.     

用激光共聚显微镜研究菌斑生物膜的结构

目的：研究菌斑生物膜的结构。方法：从牙齿表面获得完整的菌斑生物膜标本,运用激光共聚焦显微镜对其进行断层扫描和分析。结果：菌斑生物膜是由分布不均匀的细胞、基质和空隙构成的复杂结构。结论：应用激光共聚焦显微镜技术研究菌斑生物结构是一种可行的方法。     

Fluoride-induced precipitates on enamel surface and subsurface areas visualised by electron microscopy and confocal laser scanning microscopy

The present study examined the enamel surface after in vitro topical treatments with a neutral 2% NaF solution. For minimising the risk of artefacts, samples were inspected without pre-treatment as fresh, naturally wet specimens by complementary techniques: variable pressure electron microscopy (VP-SEM) and confocal laser scanning microscopy (CLSM). VP-SEM provided information on the surface morphology, whereas CLSM allowed nondestructive visualisation of subsurface areas. Neutral NaF solutions induced globular precipitates on the enamel surfaces. If the globules formed may be described as "calcium fluoride like material", the additional information of this experiment is that, after interaction with neutral solutions, they also contain considerable amounts of NaF. When the NaF solutions were soaked on pellets and then were brought into contact with the enamel surfaces, NaF crystallites of cubic shape are formed. Confocal optical tomographies of subsurface enamel after treatments with neutral NaF solutions revealed partly coroded enamel structures, whereas VP-SEM showed intact surfaces. In between the coroded. areas, a fine granulate precipitate could be observed. This is evidence that fluoride induces the formation of sub-surface precipitates only when applied during demineralisation. The precipitate could be readily removed by 24-h contact with a KOH solution.     

Three-dimensional topographic and metrologic evaluation of dental implants by confocal laser scanning microscopy

Background: Surface topography of dental implants has changed during the past few years; however, the last systematic study on this topic is dated 1993. Purpose: The aim of this study was to correlate dental implants by surface analysis. Materials and Methods: A microtopographic analysis of 35 dental implants was performed using confocal laser scanning microscopy. Roughness value (S_a) and developed surface area (ratio, S_dr) were calculated. Implants were grouped according to their surface treatment: “minimally rough” with no further surface treatment (n= 2); ablative structured using etching or blasting (n= 17); titanium plasma spray coated (TPS; n= 9); coated with hydroxyapatite (HA; n ‐ 7). Results: Most implants (n= 16) showed S_a values between 3.0 and 5.0 um. The developed surface area has a mean value of 13.5 and an SD of 6.52. Minimally rough implant surfaces show the lowest S_a values (mean 0.5 um). Implants with ablative surface treatment have mean S_a values of 3.1 m. Both groups with additive surface treatment (TPS and HA) present similar roughness values with a mean of 6.0 um and 5.8 um, respectively. Ratio Sdr ranges from 3.1 for the minimally rough implants to 11.4 for the ablative treated implants and 14.3 for TPS‐coated and 18.4 for HA‐coated implants. There is a significant difference between the roughness and ratio values of the different groups. The topographic images show a typical surface according to the underlying surface treatment. Conclusions: We can confirm the “classic” grouping of dental implants by type of surface treatment into the groups minimally rough, ablative, TPS coated, and HA coated as these treatments lead to different ascending S_as; however, the additional value of the ratio S_dr including both spatial and amplitude aspects of the surface could not be confirmed in this study. Functional parameters describing the topographic differences are still lacking.     

Microradiography and confocal laser scanning microscopy applied to enamel lesions formed in vivo with and without fluoride varnish treatment

Abstract The aim of the present investigation was to combine 2 techniques suitable for lesion characterization: quantitative microradiography (TMR) and confocal laser scanning microscopy (CLSM) on in vivo induced lesions with and without a fluoride varnish (Duraphat®) treatment. Orthodontic bands were attached to premolars to be extracted for orthodontic reasons to induce enamel caries on the buccal surfaces. In the caries development part of the study, 1 tooth of each pair received at the start 1 topical application with the fluoride varnish Duraphat®, the contralateral tooth serving as untreated control. All teeth were extracted after 4 weeks. In the caries progression part, the premolars were banded for 4 weeks without any treatment to induce caries. 1 premolar in each pair was then extracted and analyzed. The contralateral tooth received one Duraphat® application and was extracted after another 2 weeks. In the caries development part of the study. TMR analysis showed that the Duraphat® treatment on sound enamel reduced lesion depth by 48% compared to the untreated control. In the caries progression part, no significant difference was found between the untreated teeth extracted after 4 weeks and the fluoride treated lesions extracted after another 2 weeks. CLSM images agreed well with the TMR measurements. In the fluoride treated lesions, the CLSM images showed more light scattering indicating precipitation of material.     

激光共聚焦显微镜观察不同光照模式对复合树脂充填体微渗漏的影响

目的:激光共聚焦显微镜下观察比较不同光固化模式下复合树脂固化后微渗漏的差别。方法:选取30个离体牙在颊面备4 mm×4 mm×2 mm窝洞,光固化树脂(3M Z350)分层固化充填,并随机分为A、B、C 3组,分别用弱光引导(A组)、间歇光照(B组)和高强度光照(C组)3种不同光照固化模式进行固化。标本充填完成后在37℃水浴条件下分别置于1 g/L罗丹明B荧光染料浸染,24 h后激光扫描共聚焦显微镜测量染液渗入深度,定量评价微渗漏程度,并对检测结果进行统计学分析。结果:A、B、C 3组微渗漏差异无统计学意义(P>0.05)。结论:弱光引导和间歇光照技术对复合树脂的边缘封闭性无明显影响。     

A scanning electron and confocal laser microscope investigation of tetracycline-affected human dentine

Because of the well reported dental side-effects of tetracycline administration, the drug should not be administered to children. However, it and its derivatives are often administered over a prolonged period for treatment of acne in young adults. Dental side effects are also noticed in these patients. The aim of this study was to examine with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) the root dentine of a tooth from a young adult affected by tetracycline therapy. The palatal root of an upper third molar, demonstrating distinct areas of tetracycline staining, was removed from the crown and sectioned longitudinally to produce two samples. The pulp tissue was peeled off the dentine and any remaining non-mineralized tissues were removed with sodium hypochlorite. One sample was prepared for SEM and the other as a ground section which was examined with a CLSM. It was demonstrated by SEM that the mineralizing front of unaffected dentine was of a normal calcospherite appearance; in contrast, the mineralizing front of the tetracycline-affected dentine was devoid of calcospherite formation and many surface defects were apparent. In addition, the number of dentinal tubules associated with the defects was reduced. It was shown by CLSM that tetracycline bands were made up of numerous smaller bands and that peritubular dentine not associated with fluorescent bands had incorporated tetracycline. The CLSM examination of the mineralization front of the affected dentine revealed that fluorescence of dentine was restricted to the peritubular dentine. The results confirm that dentine mineralization is affected by systemic tetracycline therapy and that tetracycline can be incorporated into peritubular dentine after mineralization of the primary dentine matrix.     

Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans

The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre‐operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non‐invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non‐invasively.     

口腔移植材料与成骨细胞相容性的激光共聚焦显微镜观察

目的:观察不同处理的钛金属表面与成骨细胞的生物相容性.方法:利用共聚焦显微镜荧光信号通过物镜返回光电倍增管成像的原理,获取不透光的钛金属表面图像,对钛金属表面(打磨、喷砂、喷砂酸蚀表面)接种的成骨细胞骨架进行荧光标记,并用共聚焦显微镜获取荧光图像,观察细胞和钛金属表面的生物相容性,并且通过逐渐深入的多层扫描,探索细胞和移植材料结合的进一步信息.结果:喷砂表面适合成骨细胞的贴附和生长.结论: 钛金属与成骨细胞的结合情况主要与金属表面的物理形态有关.     

A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy

Introduction

The present study was designed to develop a standardized model for quantification of the effectiveness of dentin disinfection by different antibacterial solutions including a new root canal irrigant, Qmix.

Methods

Dentinal tubules from the root canal side in semicylindrical dentin specimens were infected with Enterococcus faecalis by centrifugation of the bacterial suspension into the tubules. Scanning electron microscopy (SEM) was used to verify the presence of bacteria in dentin. The outer side of dentin pieces was closed, and the specimens were subjected to 1-minute and 3-minute exposure to sterile water, 1%, 2%, 6% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Qmix. Confocal laser scanning microscopy (CLSM) and viability staining were used to quantitatively analyze the proportions of dead and live bacteria inside dentin.

Results

A heavy invasion by E. faecalis was detected by both SEM and CLSM throughout the dentinal tubules. The amount of dead cells in dentin increased with increasing NaOCl concentration and time of exposure (P < .05). Qmix was equally effective in killing bacteria in dentin as 6% NaOCl; more than 40% and 60% of the bacteria were killed by both at 1 minute and 3 minutes, respectively. One percent and 2% NaOCl and 2% CHX killed 20%–30% and 30%–40% bacteria after 1 and 3 minutes of exposure, respectively, with no statistically significant differences among the 3 agents (P > .05). In the control group, which was treated with sterile water, only 4%–6% of the bacteria were dead.

Conclusions

Centrifugation helped to create a heavy, evenly distributed infection deep into the dentinal tubules. The new model made it possible to compare the effectiveness of several disinfecting solutions in killing bacteria inside dentin by a noninvasive CLSM method.     

龋病病因相关因素的激光扫描共聚焦显微镜研究

牙菌斑生物膜作为细菌生长的微环境,在龋病形成和发展中起重要作用;口腔环境中相关因素如口腔黏膜中钙离子含量等与龋病也有一定关系。利用新兴的激光扫描共聚焦显微镜对菌斑生物膜及其他相关因素进行研究,取得了一系列新的进展,本文就此领域的相关问题作一综述。     

Efficacy of different irrigation methods on dentinal tubule penetration of Chlorhexidine,QMix and Irritrol: A confocal laser scanning microscopy study

The aim of this in vitro study was to compare the percentage of penetration of three final irrigant into the dentinal tubules after application of several irrigation techniques. Mandibular premolars were divided into nine groups (n:10): chlorhexidine (CHX) + conventional syringe irrigation (CI), CHX + EndoActivator (SI), CHX + Er,Cr:YSGG laser (LAI), QMix + CI, QMix + SI, QMix + LAI, Irritrol + CI, Irritrol + SI and Irritrol + LAI. Following irrigation, all roots were horizontally sectioned at 2 mm and 5 mm from the apex and examined using a confocal laser scanning microscopy. QMix exhibited a significantly higher penetration percentage than CHX at the apical section (P < 0.05). In the Irritrol group, SI displayed a significantly higher penetration area than CI in the middle section. Statistically significant differences were also determined between middle and apical section (P < 0.05). Use of QMix with different agitation technique seems advantageous in dentinal tubule penetration.     

钛种植体-骨间隙愈合界面成骨方式的激光共聚焦显微镜观察

目的：观察钛种植体一骨界面的新生骨形成情况,探讨在间隙愈合模型中种植体和骨表面是否存在双向成骨现象。方法：将4枚带有环形凹槽的纯钛种植体经喷砂、酸蚀处理后,植入兔股骨远端髁突内。术后第5d和第19d分别肌肉注射钙黄绿素和茜素红。采用激光共聚焦显微镜观察带种植体的硬组织切片。结果：激光共聚焦显微镜显示,种植体的间隙区域均有新生骨组织生成。在间隙区域,骨创面和相应的种植体表面分别存在绿色荧光带（钙黄绿素）,二者问不连续,而在19d时注射的茜素红所标记的红色荧光将二者连接融合,并可见大量红色荧光分布于绿色荧光周围。结论：种植体一骨界面存在远端成骨和接触成骨2种成骨方式,二者相向成骨。