We thank Faekah Gohar for proofreading the manuscript. We thank Sonja Dufentester and Elke Weber for expert technical assistance.

所有的动物实验是由根据德国动物保护法的地区伦理委员会（Landesamt献给NATUR，UMWELT UND Verbraucherschutz，LANUV，德国）的批准。明斯特大学的当地伦理委员会批准的组织学和显微镜分析使用人体组织。 1.动物与材料<ol><li>根据当地的动物保护立法，使用女性或体重20〜25克所需的DSS敏感菌株的雄性小鼠和房子。啮齿动物提供特殊的食物和饮用水灭菌水随意 。 </li><li>在高压灭菌自来水5天:通过给予3％w / v的葡聚糖硫酸钠（36,000-50,000达DSS，分子量）诱发急性 DSS结肠炎。 注:DSS的效力是高度可变取决于制造商和批次。第一个测试你的供应商提供的DSS疾病活动的诱导，为此，达ILY体重是一个可靠和客观的指标。 </li><li>结肠组织样本的组织学评价，在实验结束时安乐死用CO 2吹入（或由国家和机构准则指定）的小鼠。 </li></ol> 2.实验设置为DSS-结肠炎和体外伤口愈合测定<ol><li>细胞培养和建立伤口愈合测定。 <ol><li>生长的Caco-2细胞在95％湿度和5％CO 2的环境中，在37℃。使用RPMI培养基含有10％胎牛血清（FBS）和1％青霉素/链霉素。 </li><li>种子Caco-2细胞以4×10 5个细胞/ cm 2的在35 mm培养皿高培养插入的密度（参见图4A）。 注意:插入生成与待分析代表受伤区域中的规定的小单元的自由空间分离的两个小区覆盖的区域。 </li><li> seedin两天后变介质G。通过首先通过使用自动移液管抽吸以细胞碎片残留介质执行，冲洗用100μl磷酸盐缓冲盐水（PBS）中，并添加新鲜RPMI培养基或血清剥夺介质。 </li><li>培养的细胞在补充有20纳克的EGF /毫升血清或2微克丝裂霉素C /毫升血清，以检测在伤口愈合的行为改变血清剥夺培养基（0.1％FBS）24小时。加正常RPMI培养基对照细胞24小时。 </li><li>在培养24小时，取出，如步骤3.4中所述丢弃培养插入并执行DHM。 </li></ol></li><li>急性的DSS诱发结肠炎<ol><li>溶解在100毫升高压灭菌水以获得3％（​​重量/体积）的DSS溶液3克DSS的。提供到位饮用水的小鼠随意持续7天的该溶液中。计算溶于5ml每小鼠/天DSS溶液。无DSS的对照组小鼠自由采食提供高压灭菌的水。 </li></ol></li><li>鼠和人结肠的低温薄样切片部分的制备<ol><li>在实验结束时安乐死的CO 2吹入的小鼠。 </li><li>开腹23解剖动物的腹部。除去全结肠仔细使用镊子和手术用剪刀剪回肠和直肠末端。从盲肠到直肠末用剪刀剪结肠纵向打开的结肠。使用镊子，然后用PBS洗涤24试样取出所有的粪便。 </li><li>通过卷起从盲肠纵向芽直肠结束与向内弯曲粘膜棉花全结肠准备瑞士卷。在最佳切割温度（OCT）化合物嵌入结肠样品并在-80℃下直至进一步使用保持冷冻。 </li><li>从在最佳切割温度OCT化合物手术标本嵌入人结肠组织和在-80℃直至进一步使用保持冷冻。 </li><li> 7微米厚的OCT-化合物包埋标本的切断面与cryoto的帮助我只是之前的检查。 注:最佳样品厚度取决于持久性和所研究的组织类型的散射特性。对于使用结肠组织，层厚在噪声&gt;10μm的原因显著增加由于在定量DHM相衬图像的光散射所描述的实验中，而厚度的样品&lt;5微米显示用于从深冷切削工艺损伤诱导的伪影的风险较高。 </li><li>转移到一个玻璃对象载体滑动部分。 </li></ol></li></ol> 3.技术设备，软件和程序的数字全息图的采集和评估<ol><li>定量细胞和组织成像数字全息显微镜<ol><li>使用离轴马赫-策德尔数字全息显微术系统，用于活细胞成像25， 如图1。确保在显微镜配备有10X显微镜透镜，与玻璃对象载体幻灯片和培养皿的直径为35毫米的支架显微镜阶段，加热室保持在37℃的生理温度和软件进行定量相成像25。 注意:例如，如在肯珀等人 26和Langehanenberg 等 27描述。 注:另外，使用类似的系统，该系统能够执行亮视野显微镜和活细胞的定量相成像和解剖组织幻灯片。 </li><li>干净的显微镜透镜，用镜头清洁纸和推荐的显微镜的制造商，以除去灰尘或其它污染物的清洗剂（ 例如，乙醇）冷凝器。 </li><li>启动DHM显微镜的图像采集软件，选择"开"白光照明"明场"成像模式和切换。确保科勒 - 光量所推荐的显微镜的制造商同时观察在图像采集软件（或者标准图像采集软件可以在这个步骤中使用）的实时成像窗口的样品的样品的通货膨胀。 注:图像强度应均匀地分布在视场分布和样本位置不应该与显微镜的聚焦驱动光再聚焦过程中移动。 </li><li>选择"DHM"摄像模式时，关闭"关闭"的白光照明和开关"接通"的激光。检查用激光照射是均匀的（ 即 ，光强均匀分布在DHM显微镜的成像采集软件的实时成像窗口），并观察到离轴载波干涉条纹图案出现在适当的对比拍摄的图像（数字全息图）。 </li></ol></li><li>成像冰冻切片的制备与DHM <ol><li>取样品（低温恒温器部分的玻璃物体的载体上，厚度为7μm，如在2.3中描述）从冷冻的。解冻在室温和常压下约5分钟的样品。 </li><li>添加50 - 100微升磷酸盐缓冲盐水（PBS），为包埋介质上使用移液管，直到它完全与缓冲覆盖的组织切片。用干净的玻璃盖玻片（玻璃厚度170微米）的样品。 注:过干燥可诱导和折射率的显著变化试样的散射特性。 </li><li>确保玻璃载体和盖玻片的底部从灰尘和其他污染可诱导的光散射清洗。样品是准备与明场显微镜和DHM调查。 </li></ol></li><li>与DHM组织切片的定量相成像<ol><li>交换机上的数字全息显微镜，选择成像10X显微镜镜头。开始我在DHM显微镜法师采集软件，选择明亮的文件成像模式。 </li><li>放置组织滑如在显微​​镜载玻片支架在2中所描述），朝向显微镜物镜盖玻片。 </li><li>开关"关于"DHM显微镜明视场照明。将样品与显微镜载物台的位置，并确保感兴趣的组织区域是在实时监控窗口可见。提高使用显微镜的聚焦驱动的图像的清晰度。 注意:除了感兴趣的区域，滑动的无组织的区域也应存在于视场中。 </li><li>捕获使用图像采集软件的重点突出样品的亮场图像。 </li><li>选择"DHM"摄像模式时，关闭"关闭"的白光照明和转"上"的激光照射。选择低于3毫秒的"曝光时间"，全息记录，观察holographic离轴干涉条纹出现与在成像采集软件的实时成像窗口足够的对比度和捕获的数字全息图。 </li><li>直到亮场图像和不同的样本区域的数字全息图的足够数量的已经记录3.3.5 - 重复步骤3.3.3。全息收购现已完成。 </li></ol></li><li>伤口愈合测定为DHM成像的制备<ol><li>打开约1 DHM显微镜的培养皿加热腔 - 在实验开始前3小时，以确保在DHM测量稳定的温度条件。 </li><li>备的工作台上所需要的设备（培养皿中2.3中描述了伤口愈合测定）:移液管，镊，玻璃盖为培养皿，4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲的细胞培养基用生理温度（37℃）在无菌环境中的样品制备。 注意: </stro纳克&gt; Dulbecco氏改良的Eagle培养基（DMEM）是由10％胎牛犊血清（FCS），20毫米的HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸），和850毫克/升碳酸氢钠的。 </li><li>从培养皿中取出的塑料盖并用移液器除去细胞培养基。删除用镊子培养皿底部插入。 </li><li>用1ml的HEPES缓冲的细胞培养液2次，以去除死细胞，并在伤口区域剩余细胞组分（ 例如 ，血清） -洗样品1。加2ml HEPES缓冲的细胞培养液中，并盖上培养皿的玻璃盖。 </li><li>确保玻璃盖和培养皿底部已经从灰尘和其他污染清洗。样品是准备与DHM定时观察。 </li></ol></li><li>伤口愈合的连续多峰监测在体外用DHM <ol><li>交换机上的DHM显微镜1的培养皿加热室 - 前3小时在实验的开始，以确保在DHM测量稳定的温度条件。 </li><li>开关"的"数字全息显微镜，选择成像10X显微镜镜头。启动DHM显微镜的图像采集软件，选择"亮场"成像模式。确保，培养皿加热室是经营生理温度（37℃）。 </li><li>放置培养皿与伤口愈合测定法，如在4所述制备），在DHM显微镜的加热室。 </li><li>选择明场成像模式以及同时在DHM显微镜的图像采集软件的实时监控窗口观察它的样品与显微镜阶段的位置。观察样品的所需区域在白光照明下出现清晰地聚焦。 </li><li>捕捉白下样品的不同区域的亮场图像（伤口面积和融合细胞周边地区）光照射与图像采集软件和文件的外观，细胞密度和均匀性。 </li><li>选择DHM显微镜"明场"成像模式。选择下与DHM显微镜的图像采集软件实​​时监测窗口白光照明的合适的伤口面积。确保卷绕区域是从死细胞和无血清遗体自由，并确保双方包括单个同质细胞层，优选具有直边界。 </li><li>捕获与DHM显微镜的图像采集软件明场成像模式下的初始伤口面积的白色光图像。 </li><li>关闭"关闭"白光照明，选择"对"激光照排"DHM"模式，切换。选择低于3毫秒，全息记录的曝光时间（观察到全息离轴干涉条纹出现在T的图像采集软件的实时成像窗口足够的对比他DHM显微镜），捕捉数字全息图。 </li><li>捕捉DHM模式与图像采集软件样品全息图和重建的DHM显微镜的重建软件定量相位图像，以检查图像质量。 </li><li>选择一个合适的时间延迟-时间推移全息采集与DHM显微镜的图像采集软件（ 例如 ，3 5分钟）。 </li><li>选择该图像采集软件，其中样品与激光全息采集期间仅照亮的时间推移采集模式。 </li><li>启动伤口愈合测定法的时间推移DHM观察。 </li><li>停止时间推移收购预期的时间后，选择明场成像模式和记录样品在白光下成像的最终外观。 </li></ol></li><li>重建解剖组织的数字全息图，并确定平均折射率作为参数量化组织密度<ol><li>从与DHM显微镜， 例如软件解剖组织的数字全息重建量化相位的图像，如在肯珀等人 26和Langehanenberg 等人 27所述。 </li><li>确定在利益（投资回报）19选择适当的区域不同的组织层（上皮，粘膜下层，基质）的平均相差Δφ。 </li><li>通过使用折射计或者可选地通过使用从文献适当的值确定所述灌封介质的折射率。 （为典型的嵌入媒体的折射率值:N 水 = 1.334 28，正磷酸盐缓冲盐水（PBS）= 1.337 29中，n 细胞培养基 = 1.337-1.339 29,30）。 </li><li>计算不同组织的层的折射率从平均相衬值19 <img alt="式（1）"SRC ="/文件/ ftp_upload / 54460 / 54460eq1.jpg"/&gt;（1） 注意:在公式。 1λ是激光光（这里:λ= 532纳米）的波长，D的解剖组织的厚度（这里:7微米）和正 介质是包埋介质（此处的折射率:N 介质 = 正 PBS = 1.337，由阿贝-折射测定）。 </li></ol></li><li>重建并从时间推移伤口愈合系列观察评估数字全息图<ol><li>从与DHM显微镜软件26,27伤口愈合的观察过程中获得的时间间隔全息系列重建定量相图像。 </li><li>每个系列的定量相图像的规范化与最大相衬图像。 </li><li>确定面积S C，它是由细胞中由图像分割定量DHM相位图像所覆盖，其可以是每使用免费的软件单元探查形成（www.cellprofiler.org 31）。 </li><li>计算面积S c中的细胞Δφ 细胞的平均相衬。 </li><li>检索来自平均相差Δφ 细胞的细胞干块DM中的面积S C 15 <img alt="公式（2）" src="/files/ftp_upload/54460/54460eq2.jpg" /> （2） 注:在等式2中的DM表示蜂窝干质量，S C为是受细胞和α= 0.002米2 /千克的占有面积。 </li><li>确定积分蜂窝折射率n 细胞和细胞培养基Ñ 介质的折射率。从悬浮细胞确定ñ 细胞实验分别在30描述测量用折射ñ 网上平台 。爱特纳tively使用文献值n个 单元 30和N 中 29,30。 </li><li>从λ，Δφ 细胞 中，n 细胞和n 介质 26,32计算平均电池厚度d 细胞 <img alt="公式3" src="/files/ftp_upload/54460/54460eq3.jpg" /> （3） 注意:在公式。 3参数d 小区是平均单元厚度，λ表示激光的光的波长，Δφ 细胞表示平均相位相反，且n 细胞和n 介质是积分蜂窝折射率和周围介质的折射率。 </li></ol></li></ol>

The authors have nothing to disclose.

我们证明DHM提供了在小鼠模型结肠炎组织损伤和人体结肠组织样本体外的准确评估。此外，我们还显示DHM可连续监测上皮伤口愈合，而同时提供有关细胞改变多式联运信息。在DHM，以数字32进行捕获数字全息图的重建。因此，在比较明亮的视野显微镜，泽尼克相差和差分干涉相差显微术，DHM提供任选的随后数值焦点校正（多聚焦成像）定量相衬27。定量相位成像是基?...

炎性肠病（IBD）， 即，溃疡性结肠炎（UC）和克罗恩氏病（CD）是胃肠道1的特发性炎性病症。研究IBD的病理生理学和潜在的新药物或新的诊断方法评价特别重要性。在这两个基础研究和IBD患者的临床管理，肠黏膜已成为人们关注2,3的焦点。粘膜表示解剖边界，在该共生细菌，上皮细胞和肠道免疫系统的各种细胞成分之间的交互编排肠道动态平衡4,5。然而，在IBD患者，不受控制的和持久性肠炎症导致粘膜损伤，如溃疡或狭窄，其可以在上皮的屏障功能，它本身加剧局部炎症6的击穿终于达到高潮检测的。 <p类="jove_content"&gt;上皮伤口愈合因此，以下炎症至关重要的上皮再生，但也是胃肠道溃疡或吻合口瘘的胃肠道手术后7愈合的核心需求。上皮伤口愈合可以在体外伤口进行模拟愈合测定法和在肠道炎症8,9的小鼠模型在体外 以及 体内的方法有缺点，它限制了实验评估的准确性， 在体外测定，如古典划痕测定法，需要旷日持久的染色步骤或转染荧光发色团。他们往往可以通过细胞增殖和迁移不能被自动化10的不连续的监测的限制。 在体内模型，例如硫酸葡聚糖钠（DSS）诱发的结肠炎，经常由于看到的显著变化缺乏鲁棒读奏，在部分在实验室指标，MA不当国王这样的标记，以评估严重结肠炎11,12。发炎粘膜的组织学分析是目前仍然确定结肠炎的严重性的最有效的方法，但是这一点，像在体外上皮伤口愈合测定法，要求染色和依赖于研究者的专门知识13。 最近的数字全息显微术（DHM），定量相显微镜14的变型中，被确定为在体外和体内 15上皮伤口愈合的评价有用的工具。DHM允许组织密度的评估通过测量光路长度的延迟（OPD） ，其前景新型癌症诊断和16-18炎症相关组织改建19的定量。此外，DHM允许细胞形态动力学监测通过确定单元厚度，细胞覆盖的表面区域和细胞内（蛋白质）含量的数量<sup&gt; 15,20。 在体外试验中，DHM还使生理过程， 例如，通过评估在细胞体积和厚度21,22更改蜂窝透水性的分析。此外，DHM测量可以自动其防止研究者相关样本偏差。 在这里，我们证明在肠道炎症的小鼠模型中使用DHM的，并且也适用DHM到人体组织样品的分析为定量监测伤口愈合的作为无标记的体外测定。首先，我们评估在从与IBD的人类结肠炎小鼠组织切片的不同结肠壁层的炎症性变化。描述DHM定量相成像过程后，我们提供了使用显微镜成分，组织切片的准备和还描述了获得定量的相位图像的评价的详细说明。 下一步，我们表明，DHM可以UTIlized 体外连续多监测上皮伤口愈合，并描述像细胞层的厚度，干重和细胞体积的细胞特征的分析深入了解药物引起和生理细胞改变。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Azoxymethane (AOM)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>A5486</td><td></td></tr><tr><td>Cell Culture Flask</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>658170</td><td></td></tr><tr><td>Costar Stripette</td><td>Corning Inc., New York, USA</td><td>4488</td><td></td></tr><tr><td>Dextran sulphate sodium (DSS)</td><td>TdB Consulatancy, Uppsala, Sweden</td><td>DB001</td><td></td></tr><tr><td>DMEM/Ham&#039;s F12</td><td>PAA Laboratories - Pasching - Austria</td><td>E15-813</td><td></td></tr><tr><td>EGF</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>SPR3196</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>E 9884</td><td></td></tr><tr><td>Falcon Tube 50 ml</td><td>BD Biosciences, Erembodegem, Belgium</td><td>352070</td><td></td></tr><tr><td>Isopentane (2-Methylbutane)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>M32631-1L</td><td></td></tr><tr><td>Methylene blue</td><td>Merck, Darmstadt, Germany</td><td>1159430025</td><td></td></tr><tr><td>Mitomycin C</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>M4287</td><td></td></tr><tr><td>Microscope Slides</td><td>G. Menzel, Braunschweig, Germany</td><td>J1800AMNZ</td><td></td></tr><tr><td>O.C.T. Tissue Tek compound&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sakura, Zoeterwonde, Netherlands</td><td>4583</td><td></td></tr><tr><td>Pen/Strep/Amphotericin B</td><td>Lonza, Verviers, Belgium</td><td>1558</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Lonza, Verviers, Belgium</td><td>4629</td><td></td></tr><tr><td>RPMI 1640</td><td>Lonza, Verviers, Belgium</td><td>3626</td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td>Braun, Melsungen, Germany</td><td>5/12211095/0411</td><td></td></tr><tr><td>Standard diet</td><td>Altromin, Lage, Germany</td><td>1320</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, Leiden, Netherlands</td><td>4566</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Lonza, Verviers, Belgium</td><td>7815</td><td></td></tr><tr><td>Vitro &amp;ndash; Clud</td><td>R. Langenbrinck, Teningen, Germany</td><td>04-0002&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;&amp;micro;-Dish 35 mm with Culture-Insert, high</td><td>ibidi GmbH, Munich, Germany</td><td>81176</td><td></td></tr><tr><td>DIC Lid for &amp;micro;-Dishes, with a glass insert</td><td>ibidi GmbH, Munich, Germany</td><td>80050</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MICROM HM550</td><td>Thermo Fisher Scientific, Inc., Waltham, USA</td><td>46320</td><td></td></tr><tr><td>&lt;strong&gt;Digital holographic microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Component&lt;/strong&gt;</td><td>&lt;strong&gt;Model&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td></td></tr><tr><td>Inverted Microscope</td><td>iMIC</td><td>Till Photonics, Graefelfing, Germany</td><td></td></tr><tr><td>Laser</td><td>Compass 315M</td><td>Coherent GmbH, Luebeck, Germany</td><td></td></tr><tr><td>Microscope lens</td><td>Zeiss EC Plan Neofluar 10x/0.3</td><td>Zeiss, Goettingen, Germany</td><td></td></tr><tr><td>CCD camera</td><td>DMK 41BF02</td><td>The Imaging Source, Bremen, Germany</td><td></td></tr></tbody></table>

multimodal quantitative phase imaging with digital holographic microscopy accurately assesses intestinal inflammation and epithelial wound healing

炎性肠疾病， 即的发病率，克罗恩病和溃疡性结肠炎，已显著在过去十年中增加。 IBD的病因仍然是未知的和当前的治疗策略是基于对免疫系统的非特异性抑制。中专门针对肠道炎症和上皮伤口愈合能显著改善IBD的管理治疗的发展，但是这需要的炎性改变精确检测。目前，潜在候选药物所使用的动物模型在体内或在体外细胞培养为基础的技术，通常进行评价。组织学检查通常需要的细胞或感兴趣的组织被染色，其可以改变样本特征，此外，研究结果的解释可以通过调查专门知识而异。数字全息显微镜（DHM），根据该检测光路长度的延迟，使得染色无定量相衬成像。这使得结果与绝对生物物理参数直接相关。我们展示了如何在与DHM组织密度的变化的测定中，根据测定折射率，可以量化炎症改变，而不染色，在从小鼠和人类与结肠炎结肠组织标本的不同的层。此外，我们表现出体外 ，可能通过简单的自动测定受伤面积和形态学参数的同时测定使用上皮DHM伤口愈合的连续多无标签的监测，如干燥质量和迁移细胞层的厚度。总之，DHM表示用于肠道炎症与参数可能绝对值的评估有价值的，新的和定量的工具， 在体外上皮创伤愈合的简化量化，因此具有用于平移诊断ù高电位本身。

典型设置为DHM成像数字全息显微镜（DHM） 执行明场成像和定量DHM相衬成像，我们应用倒置显微镜如在图 1B中描绘。该系统是通过将一个DHM模块改性，如前面25所描述。通过光照生成具有倍频Nd的光的样品的数字全息图:在马赫-曾德尔配置（ 图 1</strong...

Watch this Scientific Journal Video about Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing at JoVE.com

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

multimodal-quantitative-phase-imaging-with-digital-holographic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.3K 次观看.  University Hospital Münster. Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro. 

视频: Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜:测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

科研

生物工程

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

医学

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

免疫与感染

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

生物学

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...