这项工作是支持的格拉茨医科大学生全宗 。所有受试者的知情同意。从格拉茨医科大学的研究协议的机构获得批准的。

1。标本采集<ol><li>口腔服务的4个月后取出固定腭扩展。使用NOLA旱地系统隔离，然后删除（图1和2）的家电。 </li><li>使用消毒的钳子，手套和托盘删除扩展，而不增加污染（图3和4）。 </li><li>在Sarstedt 120毫升小瓶存储对象在-20 ° C。在24小时内，取上冰和过程实验室。 </li></ol> 2。生物膜固定<ol><li>刮去生物膜用无菌手术刀片，或收集用无菌镊子件。 </li><li>添加冰冷的4％PFA解决方案，直到样品覆盖。 </li><li>混合物孵育4℃（不冻结）3至12小时。较长的固定时间或固定温度较高，可能会导致透水性较弱的寡核苷酸探针的革兰阴性细胞的细胞信封。 </li><li>删除煤灰的解决方案，并用冰冷的1X PBS。重复此步骤2-3次为Remove残余煤灰。 </li><li>重悬样品1卷。冰冷的1X PBS和添加1卷。冰冷的96％（V / V）乙醇。 </li><li>样品存放于-20 ° C根据此协议，以固定的样本，可以存储几个月到几年。 </li></ol> 3。固定样本脱水<ol><li> 5-30μL载玻片PFA固定样本材料的应用。 </li><li> 46干℃，约15分钟或更长的时间在房间温度。解冻溶菌酶和甲酰胺。 </li><li>加入250μL溶菌酶（1mg/ml的），在室温10分钟，从而促进局部破坏细胞壁进入细胞的探针的渗透。 </li><li> DIP到50％，80％和96％（V / V）乙醇，每次3分钟的幻灯片。乙醇浓度系列的脱水作用，会瓦解的细胞膜。 </li><li>干在46℃10分钟的幻灯片。 </li></ol> 4， 在原位杂交<ol><li>准备1毫升新鲜的杂交缓冲液（见表1浓度）。在这项研究中使用的甲酰胺浓度：10％（EUBmix），20％（Bac303，POGI，MUT590）或45％（LGCmix）。 </li><li>解冻寡核苷酸探针解决方案。在冰上解冻探头应保持和避光。 </li><li>添加2μL每个探头200μL杂交缓冲液，拌匀，适用于载玻片脱水样品的混合物。 </li><li>放入一个方形培养皿中一块纸巾，其余的杂交缓冲液倒到卫生纸。 </li><li>立即水平放置幻灯片入菜和关闭的菜。在烤箱放置在46℃1-5小时（90分钟就足够了，在大多数情况下）。作为水分室盘功能，防止从幻灯片杂交液的蒸发。特别是，甲酰胺的蒸发可引起非特异性探针对非靶细胞的结合。 </li><li>准备洗涤液50毫升（见表2 concentrations）。准备洗涤液50毫升管和预热至48℃水浴中。洗涤步骤是在48 ° C。 </li><li>删除幻灯片从杂交炉盘。立即洗掉的预热洗涤液的体积小，杂交缓冲液，并转移到剩余的洗涤液的幻灯片。 </li><li>将管含有洗涤液和幻灯片，在48℃水浴孵育10-15分钟。 </li><li>从管中取出幻灯片到冰冷的DDH 2 O为2-3秒，以消除残留洗涤液浸。 </li><li>空气干燥的幻灯片尽快（建议使用压缩空气）。快速干燥会降低探针的解离。 </li><li>干幻灯片可以存储在黑暗中，几个星期没有显著损失探头赋予的荧光信号在-20 ° C。 </li></ol> 5。显微镜<ol><li>经过FISH和洗衣机，APPLY两滴antifadent密切幻灯片的左，右两端（冷冻切片应该到了这一步前加热到室温）。 </li><li>放置显微镜的盖玻片在上面，等到antifadent已蔓延在整个幻灯片。请注意，过量的antifadent显微镜图像模糊。 </li><li>配备适当的过滤器或激光器的激光扫描共聚焦显微镜下观察样本。我们使用的Leica TCS单位（HCX PL APO/63x：NA 1.2）。数据可以分析软件，如IMARIS或AMIRA。 </li><li>在antifadent嵌入的幻灯片，可以储存在4℃（不冻结）长达7天前探头赋予的荧光开始下降。另外，antifadent可以删除与DDH 2 O，和干燥的幻灯片，可以长时间储存在-20 ° C。 </li></ol> 6。代表性的成果： 刮关闭固定正畸的生物膜（图5）产生合适的薄片（图6），可直接上镜镀膜玻璃幻灯片杂交。通过这种方式，可以识别不同群体的orobiome细菌在其自然的三维环境，通过标记不同的标记的特异性探针（图7和图8）细菌的rRNA。在图7中，生物膜被染成EUBmix（绿色，所有的细菌）和LGCmix（黄色，厚壁菌门）。厚壁菌显示为绿色，因为他们是黄色和蓝色的染色。在图8中，生物膜被染成EUBmix（红色，所有的细菌），Bac303（蓝色，杆菌）和POGI（黄， 牙龈卟啉单胞菌 ）。 牙龈卟啉单胞菌是黄白色，所有三个探头绑定到其DNA和重叠结果在一个白色的信号颜色。也可以识别细菌群体之间形态上的差异（图9和10）。如图9所示，其中染色与EUBmix大型集群的球状细菌（绿色，所有的BActeria）。不同形状的口腔细菌，在图10中，球状和丝状菌可区分EUBmix（红）染色观察。此外，一个典型的蘑菇状的生物膜的结构，可以通过处理CLSM数据的三维模型（图11和<a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">12），点击这里观看影片的</a> 3D建模。 <img alt="图1" src="/files/ftp_upload/2967/2967fig1.jpg" /> 图1。腭扩张器固定在原位 。 <img alt="图2" src="/files/ftp_upload/2967/2967fig2.jpg" /> 图2。诺拉干田制。 <img alt="图3" src="/files/ftp_upload/2967/2967fig3.jpg" /> 图3。消毒钳，手套和托盘。 <img alt="图4" src="/files/ftp_upload/2967/2967fig4.jpg" /> 图4。删除扩展。 <img alt="图5" src="/files/ftp_upload/2967/2967fig5.jpg" /> 图5。刮痧用无菌手术刀生物膜。 <img alt="图6" src="/files/ftp_upload/2967/2967fig6.jpg" /> 图6。树脂薄片直接杂交到镀膜玻璃幻灯片。 <img alt="图7" src="/files/ftp_upload/2967/2967fig7.jpg" /> 图7 CLSM的形象：一个特定的细菌群体的分化。 2D 3D CLSM堆栈数据覆盖。生物膜染色EUBmix（绿色，所有的细菌）和LGCmix（黄色，厚壁菌门）。 <img alt="图8" src="/files/ftp_upload/2967/2967fig8.jpg" /> 图8 CLSM的形象：一个特定的细菌群体的分化。 2D 3D CLSM堆栈数据覆盖。生物膜沾上EUBmix（红色，所有的细菌），Bac303（蓝色，Bacteroidetes）和POGI（黄， 牙龈卟啉单胞菌 ）。 <img alt="图9" src="/files/ftp_upload/2967/2967fig9.jpg" /> 图9 CLSM图像：具体形态分化。 2D 3D CLSM堆栈数据覆盖。 EUBmix沾上生物膜（绿色，所有的细菌）。球状​​细菌的大型集群（箭头）。 <img alt="图10" src="/files/ftp_upload/2967/2967fig10.jpg" /> 图10 CLSM图像：具体形态的分化。 2D 3D CLSM堆栈数据覆盖。 EUBmix生物膜染色（红色，所有的细菌）。球形（箭头低于）和丝状菌（箭头上述）可以区分。 <img alt="图11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 图11。食用菌结构，从下面的三维视图。叠生物膜薄片（关闭矫治表面刮）EUBmix和proce染色，ssed与IMARIS（CLSM图像）。 <img alt="图11" src="/files/ftp_upload/2967/2967fig11.jpg" /> 图12：蘑菇结构，3D侧视图。堆栈的生物膜薄片加工IMARIS（CLSM图像）（关闭矫治表面刮）。 <table border="1"><tbody><tr><td colspan="4"> 杂交缓冲液（200微升） </td></tr><tr><td> 甲酰胺浓度 </td><td> 10％ </td><td> 20％ </td><td> 45％ </td></tr><tr><td> 5 M氯化钠 </td><td> 36 </td><td> 36 </td><td> 36 </td></tr><tr><td> 1米的Tris - HCl </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 2％SDS </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> 足总杯 </td><td> 20 </td><td> 40 </td><td> 90 </td></tr><tr><td><strong&gt; DDH 2 O </td><td> 138 </td><td> 118 </td><td> 68 </td></tr><tr><td> 任何鱼探头 </td><td> 2 </td><td> 2 </td><td> 2 </td></tr></tbody></table> 见表1。杂交缓冲液成分（μL浓度）。 <table border="1"><tbody><tr><td colspan="4"> 洗涤缓冲液（50毫升） </td></tr><tr><td> 甲酰胺浓度 </td><td> 10％ </td><td> 20％ </td><td> 45％ </td></tr><tr><td> 5 M氯化钠 </td><td> 4500 </td><td> 2150 </td><td> 300 </td></tr><tr><td> 1米的Tris - HCl </td><td> 1000 </td><td> 1000 </td><td> 1000 </td></tr><tr><td> 0.5 M EDTA </td><td> 0 </td><td> 500 </td><td> 500 </td></tr><tr><td> DDH 2 Ø </td><td> 44 500 </td><td> 46 350 </td&gt; <td> 48 200 </td></tr></tbody></table> 表2。成分的洗涤液（μL浓度）。 电影1 <a href="http://www.jove.com/files/ftp_upload/2967/Film1 Klug Santigli 01_2_JES_Web.mov">点击这里观看影片。</a>

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没有利益冲突的声明。

此处描述的协议是一个非常可行的方法，从硬质材料的收集染色生物膜。采样和杂交是最关键的步骤。在采样过程中，必须小心收集足够厚度的切片，以确保生物膜的结构保持不变。在杂交过程中，它是必不可少的，以避免在温度的过度波动，从而避免了非特异性的约束力，或荧光标记的探针具有约束力的损失。这种鱼的协议是一种优雅的方法，直接染色玻片上不扰乱其组成正常的异构生物膜。幻灯片可以立即用于显微镜，而不需要再次移动样品。这样一来，对改善管染色是通过被应用于生物膜的剪切力。片&gt; 50微米厚，可以以这种方式准备和调查。

<table border="1">
	<tbody>
		<tr>
			<th>Oligonucleotides</th>
			<th>Target organism</th>
			<th>Fluorochrome</th>
		</tr>
		<tr>
			<td>EUB338 - 338III</td>
			<td>Eubacteria</td>
			<td>Cy3</td>
		</tr>
		<tr>
			<td>LGC354 A-C</td>
			<td>Firmicutes</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>Bac303</td>
			<td>Bacteroidetes</td>
			<td>Cy5</td>
		</tr>
		<tr>
			<td>Mut590</td>
			<td>Streptococcus mutans</td>
			<td>FITC</td>
		</tr>
		<tr>
			<td>POGI</td>
			<td>Porphyromonas gingivalis</td>
			<td>FITC</td>
		</tr>
	</tbody>
</table>
Table 3. Oligonucleotides (refer to probeBase13 for details).
<table border="1">
	<tbody>
		<tr>
			<th>Component</th>
			<th>Company</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>&nbsp;</td>
			<td>4% solution in PBS</td>
		</tr>
		<tr>
			<td>1 x PBS</td>
			<td>&nbsp;</td>
			<td>3 x solution prepared</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Millipore</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (100%)</td>
			<td>Merck</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Roth</td>
			<td>100 mg/ml stock solution</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Roth</td>
			<td>99.5% solution</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2% SDS</td>
			<td>Roth</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FISH probes (oligonucleotides)</td>
			<td>Invitrogen, Sigma</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold antifadent</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<th>Equipment</th>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>IKA Tez</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Sarstedt</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polysine-coated slides</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 4. Materials and equipment.

oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy

天然异构生物膜共聚焦激光扫描显微镜（CLSM）今天是促进全方位的染色技术，他们在荧光原位杂交（FISH）。1,2，我们进行了试验研究，在口腔生物膜样品收集固定正畸（腭扩展）染色鱼，目的是评估天然生物膜和菌斑的堆积的三维组织 。3,4鱼创造一个机会，在其本土生物膜环境染色细胞，使用荧光标记的16S rRNA的目标的探测。4-7,19相比，替代技术，如免疫荧光标记，这是一种廉价，准确，直观的标记技术，以调查在混合生物膜财团不同的细菌群体。18,20一般探头使用绑定到真细菌（ EUB338 + EUB338II + EUB338III以下EUBmix），8-10厚壁菌门（LGC354交流;以下简称LGCmix），9,10和杆菌（Bac303）11此外，特异性探针结合变形链球菌 （MUT590）12,13和牙龈卟啉 （POGI）13,14 。极端的表面材料硬度（不锈钢和丙烯酸树脂）迫使我们准备生物膜找到新的途径。由于这些表面材料不能随时切用cryotome，各种抽样方法进行了探讨，以获得完整的的口腔生物膜。这些方法最可行的是在这个沟通。生物膜的丙烯酸树脂小片被刮掉，用无菌手术刀，注意不要破坏生物膜结构。镊子是用来收集从钢铁表面的生物膜。一旦收集到的样本是固定的，直接放在polysine镀膜玻璃幻灯片。这些幻灯片直接与探头米鱼entioned以上。各种鱼类协议相结合，并修改，以创建一个新的协议，很容易处理。5,10,14,15随后共聚焦激光扫描显微镜分析样品。著名的配置3,4,16,17可以可视化，包括蘑菇式地层和渠道弥漫的球状细菌群。此外，这些典型的生物膜结构的细菌组成进行了分析和创建二维和三维图像。

Watch this Scientific Journal Video about Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy at JoVE.com

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

我们目前的协议矫治器的结构和成分分析的天然口腔生物膜与<em&gt;原位</em&gt;杂交（FISH）和共聚焦激光扫描显微镜（CLSM）。口腔生物膜样品分别采自腭扩展，刮关闭其表面的丙烯酸树脂片和指分子处理。

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

oral-biofilm-analysis-palatal-expanders-fluorescence-situ

Research

JoVE Journal

Medicine

15.9K 次观看.  Medical University of Graz. 我们目前的协议矫治器的结构和成分分析的天然口腔生物膜与原位杂交（FISH）和共聚焦激光扫描显微镜（CLSM）。口腔生物膜样品分别采自腭扩展，刮关闭其表面的丙烯酸树脂片和指分子处理。

视频: Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

科研

医学

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

口腔生物膜取样在健康儿童中的微生物分析

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

牙种植体不同材料的口腔生物膜形成

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

老年人外行人员口腔健康评估

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

使用扫描电子显微镜可视化膜褶皱形成

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

多发性骨髓瘤骨髓涂片的相间荧光 原位 杂交

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

使用口内荧光相机对牙菌斑进行半自动平面定量

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

捕获口腔粘膜下纤维化组织样本用于空间组学分析

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

作者聚焦：揭开口腔潜在恶性肿瘤的神秘面纱 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

激光捕获显微切割从口腔粘膜下纤维化样本中分离具有形态学和空间信息的细胞

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...