笔者想感谢哈维尔·加西亚·E.琳恩和对格隆克沙尔技术援助和莫雷特K. Raarup了富有成果的讨论。这项工作是由奥胡斯大学研究基金会资助并西蒙基金会探马。

实验方案进行了审查，并通过奥胡斯郡的伦理委员会（M-20100032）的批准。 1.成比例的染料的共聚焦显微镜校准<ol><li>对于图像采集，使用配有孵化器倒置共聚焦显微镜，一个63X / 1.2数值孔径的水浸泡的目的，一个543纳米激光线和一个META检测器。 </li><li>制备的HEPES缓冲液储备溶液（50mM的，以0.1个pH单位的步骤调节至pH 4.5-8.5）。吸取100μl的每种溶液成用于荧光显微镜一个透明底96孔板的孔中。 </li><li>处理比例染料C-SNARF-4时，戴上丁腈橡胶手套。制备在二甲基亚砜染料的1mM储备溶液。加入5微升原液，以每孔HEPES缓冲液。放置在显微镜的96孔板中。 </li><li>打开显微镜。打开显微镜软件。点击以下面板:采集→激光;收购→MICRO;收购→配置;收购→扫描;收购→舞台。热身孵化器至37℃。 </li><li>通过点击543 nm激光和"关于"按钮，在"激光控制"窗口打开543纳米激光线。选择在"显微镜控制"窗口中的63X / 1.2数值孔径的水浸泡的目的。 </li><li>将META探测器576- 608纳米（绿色）和629- 661纳米（红色）的时间间隔（"配置控制"→"CHS"）中同时监测荧光。调整激光功率（"配置控制"→"激励"）。设置针孔，得到1.6微米的光学切片厚度（"扫描控制"→"针孔"）。 </li><li>获得的每个的HEPES缓冲液，5微米的96孔板的玻璃底部上方的图像。注意:只要聚焦平面位于下方的玻璃底部，没有荧光可以看出屏幕上。每三个图像后，将激光功率为零，拍摄图像的背景减除。 </li><li>执行一式三份的标定实验（1.2-1.7）。 </li><li>确定在所有的红色和绿色的图像的平均荧光强度和标准偏差。 <ol><li>根据等式计算比R和平均值的标准误差，S R，对于每一个图像（1）和（2） （1） <img alt="公式1" src="/files/ftp_upload/53622/53622equation1.jpg" /> （2） <img alt="方程2" src="/files/ftp_upload/53622/53622equation2.jpg" /> 克，R，S G和S R是在各自的绿色和红色的图像的平均值和标准偏差; B G，B R，S BG和宽单峰都是为背景图像的对应值。1. <eM&gt; 2是成像像素数。 </li><li>绘制所计算的比率，用于从图中的三次重复校准实验各pH值，并从该系列的数据点的构建拟合曲线（ 即使用软件SIGMAPLOT 13）。使从可比率转换成的pH值10的拟合曲线的数学函数。 </li></ol></li></ol> 2. 在原位生长的生物膜牙样品采集<ol><li>选择履行相关的研究入选和排除标准的志愿者。使它们的上和下齿弓的藻曝光。使从这些展示铸造模型和下颚制造的丙烯酸系夹板。设计与舌正畸钢丝，使志愿者咬入正常咬合连接12颊丙烯酸法兰夹板。 </li><li>在丙烯酸夹板的颊凸缘钻头衰退（ 图1 </strong&gt;）牙科丙烯酸钻的帮助，以便收集生物膜玻璃砖的插入。凹陷的深度至少应为1.5毫米，而凹陷的宽度和长度可以根据要插入玻璃板坯的数目而变化。 </li><li>生物膜收集，使用定做的非荧光玻璃板坯（4×4×1毫米3）与砂砾1200的表面粗糙度，以模仿天然釉质11定植图案。 </li><li>通过之前安装高压灭菌玻璃砖。安装在每个侧的颊凸缘稍微凹进至丙烯酸表面的表面上的凹陷粘蜡玻璃板坯，以保护从由面颊11的移动施加剪切力的生物膜。 注意:放置在一个衰退玻璃板坯的数目可以，3和14之间变化取决于研究的目的。 </li><li>插入志愿者口设备。指示volunTEER保留器具口内整个实验期间。指示志愿者以存储在用一块湿纸巾的正畸保持器容器的器具刷牙和食物和水以外的饮料的摄取期间，在室温（保持它湿润）。指示志愿者不接触与玻璃砖颊丙烯酸法兰同时放置和移除设备。 注意:实验周期可以根据研究（一天至几周）的目的而有所不同。 </li><li>仔细实验期结束时删除该设备上的玻璃砖。用刀除去周围的板坯粘性蜡并用一对镊子的传递它们到一个密闭容器中，朝上生物膜，直到显微分析。保持与湿纸巾湿润的容器。生物膜收集后进行几个小时内pH值成像。 </li></ol> 3.生物膜的pH成像<ol><li>准备通过根据德容等人 13的方法中加入二硫苏糖醇，以收集唾液的唾液溶液。滴定唾液溶液至pH 7.0，并添加葡萄糖至0.4％（重量/体积）的浓度。每生物膜移液器将100μl要分析成用于显微镜的玻璃底96孔板中。加入5微升每孔的比例染料。 </li><li>放置在显微镜载物台96孔板中。打开显微镜和543纳米的激光线。热身孵化器至37℃。使用相同的显微镜设置作为染料的校准（见步骤1.5-1.6）。等待30分钟，直到96孔板已达到工作温度。 </li><li>拿起一个或多个玻璃砖采用了超薄一套镊子，并将其放置在唾液充满井，每口井一个平板，朝下的生物膜。 </li><li>获取单个图像（"扫描控制"→"单"）或Z-栈（"扫描控制"→"开始"）spanni纳克在不同领域的生物膜的深度。获得的z栈选择要被成像（"扫描控制"→"Z设置"→"数片"）的片数，并标记为第一，并在显微镜软件的最后切片的z位置（"扫描控制"→"Z设置"→"标记为先";"马克最后"）。 注意:Z-栈具有高达75微米的可与细胞外和细胞内区域之间的良好的对比度被收购的深度。 </li><li>要遵循的观点随着时间的推移微观领域pH值的变化，标志着显微镜软件（"第一阶段和聚焦控制"→"马克·波什"），XY位置，并在连续的时间点采取重复图像（"扫描控制"→"单"）。经常服用的图像与背景减除设置为零激光功率。 </li></ol> 4.数字图像分析<ol><li>世博RT显微图像为TIF文件，使用显微镜软件（"宏"→"文件批量导出"）的文件批量出口。标记要导出的文件，并保存红色和绿色通道图像单独的文件夹为TIF-文件（"开始批量导出"）。重命名这两个文件夹给他们的顺序编号的文件。 </li><li>导入红色和绿色图像串联成软件如daime（在微生物生态学数字图像分析）14。段与个人选定的亮度阈值（段→自动分割→自定义阈值）的绿色通道图像。小心地选择（通常是20与80之间）的亮度的阈值，以使所有的细菌（除外基质亮），但不是矩阵将被识别为分割期间的对象。验证目视即识别为对象的区域对应井到细菌生物质。 </li><li>传输分段克对象层颖通道图像对应的红色通道图像（段→传输对象层）。使用对象编辑器功能，拒绝和删除红色和绿色通道图像的所有对象。现在仅外基质被留在所述生物膜的图像。导出处理的图像系列为TIF文件。 </li><li>导入图像串联成的ImageJ（http://rsb.info.nih.gov/ij; v.1.47）。确定在与激光拍摄的背景图像的平均荧光强度断开（分析→直方图）。减去红色和绿色的图像适当背景（流程→数学→减）。 </li><li>仍然在ImageJ的，本身（过程→图像计算器）划分绿色形象系列（G1）。然后乘以所产生的图像系列（G2）的绿色形象系列（G1）。这将产生一个图象系列（G3），其中NaN被分配给属于该被确认为在daime对象区域的所有像素。继续在T他相同的方式与红色图象系列（R1 / R1 = R2，R2点¯xR1 = R3）。 注意:作为细菌的生物质从图像在步骤4.3除去，荧光强度是在这些区域0。步骤4.5是必要的值0转换为NaN时，其允许在步骤4.6比计算。 </li><li>应用&#39;是什么意思"过滤器（处理→过滤器→均值;半径:1像素），以补偿检测器噪音。除以红色图像系列（过程→图像计算器）的绿色形象系列。这导致的绿色/红​​色比在图像的外空间中的每个剩余的像素。利用虚假的着色图像中的比例图示（图像→查找表）。计算每个图像的平均比率（分析→直方图）。 </li><li>根据1.9.2下安装的功能）转​​换绿/红比值的pH值。注:校正数据和拟合曲线的例子可以在Schlafer 等可见人2015年11。 </li></ol>

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作者什么都没有透露。

相比，电极或微电极测量4-6生物膜pH为微观监测提供了若干优点。显微技术允许确定pH值以高空间分辨率和允许在不机械地扰乱生物膜捕获在生物膜水平和垂直的pH梯度。的微观pH监测先前的尝试，但是，没能在生物膜1,7,9外和细胞内pH值之间进行区分。由于细菌的动态平衡，细胞内pH从外pH不同，并且如果内和细胞外隔室向记录荧光发射的pH计算可以是无效的。使用第二荧光染料与pH?...

这里所描述的方法可以监控在牙科生物膜外pH在4.5和7之间的范围内，使用比例染料seminaphthorhodafluor-4F 5-（和-6）羧酸（C-SNARF-4）在共聚焦激光扫描显微镜组合和数字图像分析。所采用的荧光染料是pH敏感，并显示在其荧光发射取决于质子化的状态的转变。在580处的质子化分子峰的荧光发射，并且去质子分子在640nm 1的发射。荧光发射强度的两个探测窗口包括两个发射峰的比率（576 - 608 nm和629 - 661纳米）从而反映pH值在液相，不论染料浓度。用〜6.4的pK a的染料适用于适度酸性环境中的可视化的pH值。 PH值在细菌生物膜是对所有代谢过程至关重要。在牙科生物膜的情况下，pH值在细胞外基质为龋齿的发展的关键毒力因子。 pH值较低的生物膜牙接口引线过长，以减缓潜在的搪瓷2脱钙。由于生物膜，代谢物，包括有机酸的复杂的三维结构，不是均匀地在整个生物膜分布。高少产酸微环境可以紧密的空间接近3被发现。 几十年来，在生物膜垂直pH梯度进行记录微电极4-6的帮助。虽然他们提供了一个良好的空间分辨率，由于其小针尖大小，他们不是非常适合于监控水平梯度。此外，电极的插入机械干扰生物膜。荧光定量显微技术提供可视化生物膜中的不同区域的pH值的变化没有机械干扰的优势NCE。视不同的显微镜视野可以自由选择和过长时间1,7-9反复成像。然而，解译显微生物膜图像时，它的荧光从微生物生物质和荧光从细胞外空间导出导出区分是重要的。在酸性条件下，pH值的细菌细胞内是从pH值在细胞外基质不同，因为细菌在积极三磷酸腺苷10的费用运输在其细胞膜质子。在龋齿的上下文中，而低外pH导致脱矿质胞内细菌的pH不会对底层搪瓷有直接影响。在同时包含无菌区域和细菌显微图像均pH值会导致错误的结果。为了可视化的细菌的生物量和细胞内外区域之间区分使用具有pH敏感的染料沿着其它污渍带来从头出细胞外空间的荧光污染和假测量11的风险。 因此，本手稿描述了一个双重功能的比例染料;既作为pH标记，并作为通用细菌污点。作为染料是向上集中在细菌细胞中，共焦显微成像的组合和一个准确的数字图像分析过程允许在薄牙科生物膜确定4.5和7.0之间的范围外的pH值。

<table><tbody><tr><td>Zeiss LSM 510 META</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>C-Apochromat 63X water immersion objective</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>XL Incubator</td><td>PeCON</td><td>N/A</td><td></td></tr><tr><td>SNARF-4F 5-(and-6)-Carboxylic Acid</td><td>Life Technologies</td><td>S23920</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>11344-041</td><td></td></tr><tr><td>Costar 96-well black clear-bottom plate</td><td>Fisher Scientific</td><td>07-200-567</td><td></td></tr><tr><td>Custom-made glass slabs (4 x 4 x 1 mm&lt;sup&gt;3&lt;/sup&gt;; 1,200 grit)</td><td>Menzel</td><td>N/A</td><td></td></tr><tr><td>Alginate impression material</td><td>GC Corporation</td><td>N/A</td><td></td></tr><tr><td>Acrylic Adjusting Logic Sets/set of acrylic dental burs</td><td>Axis Dental</td><td>LS-906</td><td></td></tr><tr><td>Orthodontic retainer containers</td><td>Spark Medical Equipment Co., Ltd</td><td>SK-WDTC01</td><td></td></tr><tr><td>Sticky wax</td><td>Dentsply</td><td>N/A</td><td></td></tr><tr><td>Chewing paraffin wax&amp;nbsp;</td><td>Ivoclar Vivadent AG</td><td>N/A</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>D0632</td><td>Used during preparation of salivary solution</td></tr><tr><td>0.45 &amp;micro;m and 0.2 &amp;micro;m syringe filters</td><td>Sigma Aldrich</td><td>CLS431220; CLS431219&amp;nbsp;</td><td></td></tr><tr><td>daime</td><td>University of Vienna, Austria</td><td>http://dome.csb.univie.ac.at/daime</td><td></td></tr><tr><td>ImageJ</td><td>NIH, Bethesda, Maryland, USA</td><td>http://imagej.nih.gov/ij/</td><td></td></tr></tbody></table>

ratiometric imaging of extracellular ph in dental biofilms

牙齿上细菌生物膜的pH值是对于龋齿，具有高的全球流行的疾病至关重要。营养物和代谢物没有在牙科生物膜分布均匀。吸附并反应在生物膜有机物质之间复杂的相互作用降低了溶质的扩散路径，并创建活性分子的陡坡，包括有机酸，整个生物膜。定量荧光显微方法，例如荧光寿命成像或pH ratiometry，可以采用在牙科生物膜不同微环境可视化的pH值。 pH值ratiometry利用在pH敏感染料的荧光发射的pH依赖性移。在两个不同波长的发射的比例的计算允许确定在显微图像局部pH，不论染料的浓度。相反微电极的技术允许与监测实时垂直和水平的pH梯度出机械干扰生物膜。然而，必须小心以生物膜的细胞内外室之间准确区分。在这里，比例染料，seminaphthorhodafluor-4F 5-（和-6）羧酸（C-SNARF-4）被用来监测在未知物种组成的体内生长牙科生物膜的细胞外pH值。在暴露于葡萄糖的染料是向上集中在生物膜的所有细菌细胞内;因此它被用来作为一个通用细菌染色和细胞外pH值的标记。共聚焦显微图像采集后，细菌的生物量从使用数字图像分析软件，它允许以独占方式计算外pH的所有照片删除。 pH值ratiometry与比例染料是非常适合于研究高达75微米厚的薄的生物膜的细胞外pH值，但被限制在pH范围4.5至7.0之间。

所提出的方法允许监测外pH在pH范围牙科生物膜从4.5至7中的实时的不同微环境下降。如果如上述被选择的实验条件下，pH值开始于生物膜的所有领域滴暴露于葡萄糖后不久。 当pH值在生物膜滴剂，细菌细胞成为短时间（&lt;1分钟）内可见，随着比例染料在细胞（ 图2A）upconcentrated。在一开始，生物膜开始酸生产之前?...

Watch this Scientific Journal Video about Ratiometric Imaging of Extracellular pH in Dental Biofilms at JoVE.com

Ratiometric Imaging of Extracellular pH in Dental Biofilms

在口腔生物膜细胞外pH值成比例成像

pH敏感比例染料以共聚焦激光扫描显微镜和数字图像分析结合用于监测实时牙科生物膜的细胞外pH值。

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

ratiometric-imaging-of-extracellular-ph-in-dental-biofilms

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  Aarhus University.  pH敏感比例染料以共聚焦激光扫描显微镜和数字图像分析结合用于监测实时牙科生物膜的细胞外pH值。 

视频: Ratiometric Imaging of Extracellular pH in Dental Biofilms

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

科研

化学

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

免疫与感染

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

<meta charset="utf8"></head><body>实时体外监测细胞外 pH 值

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

体外 实时监测细胞外 pH 值

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

生物化学

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

在口腔生物膜细胞外pH值成比例成像

摘要

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此视频中的章节

合成，细胞和交货

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

细胞和生物膜细胞外成分的定量并发

体外实时监测细胞外 pH 值

体外实时监测细胞外 pH 值

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

在口腔生物膜细胞外pH值成比例成像

摘要

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合成，细胞和交货

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

细胞和生物膜细胞外成分的定量并发

体外 实时监测细胞外 pH 值

体外 实时监测细胞外 pH 值

通过组织的X射线激发发光化学成像对植入物相关感染进行高空间分辨率化学成像

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

体外实时监测细胞外 pH 值

体外实时监测细胞外 pH 值