这项工作是部分资金由科学教育合作伙伴奖（SEPA）R25 RR025066 2008-2012从卫生部和全国学院国家研究资源中心之间。感谢您对同事的文学，我们的结果和反馈在各种实验室场景使用DPP的深思熟虑的讨论的味道现场:海耶斯约翰，苏科尔德韦尔，保罗·布莱斯林，卡拉舒伯特。也谢谢领导自然与科学的丹佛博物馆，工作人员在品尝实验室的远征内生遗传学中支持公民科学的。最后，感谢所有我们的志愿者公民科学家为他们的时间和精力来开发和使用此协议，其中包括:熊阿拉贡，迈克·阿彻，苏阿塔曼，迈克尔·巴格利，A&#39;nette贝特朗，戴安娜博伊尔，温迪隐蔽，萨沙杜利帕蒂德雷弗，杰西卡ERN，劳拉Harmacek，肖恩希巴德，乔伊斯哈钦斯，朱马特，莱塔基恩，威廉Leenhouts，千金露kjan，阿什利·马修斯，迈克·毛瑟，斯蒂芬妮·米勒，海丽摩根 - 罗德里格斯，格里芬Scherma，贾静雯席克丹茨，特丽西门，狄兰·托马斯，鲁迪·托雷斯，泰勒·威尔逊，黛安Woltkamp。

打进本研究中的图像收集在实验室味的遗传学自然与科学（博物馆）10丹佛博物馆进行了较大的口味研究的一部分。较大的研究对象为参观者（N = 1195）从18-93不等年龄。虽然受试者来自六个七大洲，参加者主要是欧洲血统。每个主题的数据收集在一个30分钟实验课。西方机构审查委员会批准的协议，知情同意书给予和主题志愿的时间和他们参与这项研究并没有得到补偿。 1.数据收集<ol><li>图像捕捉<ol><li>显示科目如何构成自己的照片（ 图1），什么所拍摄图像的样子。 <img alt="图1" src="/files/ftp_upload/52860/52860fig1.jpg" /> 图1:图像捕捉姿势。 </str翁&gt;这是为了正确姿势舌头的照片显示，在拿到实验室科目照片。 </li><li>直接科目干自己的舌头纸手巾，离开舌头从口中伸出。 </li><li>在使用无菌，人造纤维敷贴用1英寸尖端1:36浓度到舌头的顶点应用大约3毫升蓝色食用染料。有受试者舌头返回到他们的嘴和吞咽去除多余的染料。 </li><li>有议题提出了自己作为如图1所示，用自己的双手和胳膊肘撑着稳定表的下巴。直接科目延长他们的舌头是舒适的距离，并确保它轻轻地牙齿之间。 </li><li>坚持2.5cm的一片滤纸，用10毫米直径的圆形切口冲压在其舌的左侧旁中线前的前端（参见图1）。 </li><li>取舌，CA的至少三个特写图像pturing完整的10 mm圆柱切口，以确保区域内的所有可视化的FP。使用安装在三脚架上稳定高品质的数字点和傻瓜相机的微距设置。 <ol><li>放大到整个切口拍摄，同时还相机的建议变焦范围内的程度。确保相机镜头的平面平行于舌头的平面，并且该切口出现的长圆形的照片代替。查看照片，以确保它们是高品质的和有用的计数。 </li></ol></li></ol></li><li>图像选择和准备<ol><li>将照片上传到电脑。 </li><li>采取了同一主题的舌头的图像的基础上，在舌头上最高的变焦和最不扭曲角结构之间清晰的定义作进一步的分析只选择一个，这样，舌头在宽阔平坦朝向相机。 </li><li>在开源打开所选原始图像，ftware，ImageJ的。点击"插件"，并在下拉菜单滚动到"分析"，然后单击"细胞计数"。当细胞计数打开后，点击"初始化"将图像链接到细胞计数。 注:在这里，使用ImageJ的1.45秒，32位Java 1.6.0_10版。 <ol><li>使用50％的标准放大倍率为得分之间并用于随后的步骤，以人评分的FP使用的一致性。移动此图像到屏幕的左侧旁细胞计数器。此图片将被称为复制A（见屏幕图2，左侧）。 </li></ol></li></ol></li><li>用于测量乳头的直径打开RAW图像（复印件B）的第二个副本被量化。放大，并根据需要向各个射手的整个评分处理的优先出来。移动此拷贝到屏幕右侧，这样的两个副本是不小心混淆。 （参见图2，右侧屏） </li><li>单击行工具。尽可能在必要时使用复制B，变焦，精确地绘制穿过滤纸10mm的内圆直径在任何角度，然后单击"分析"和"设置比例。"中的"已知的距离"填写为"10 "验证规模是衡量一个备用角度正确，并确保直径范围为9.8-10.2毫米之间。如果不是，重复此步骤。 </li></ol><img alt="图2" src="/files/ftp_upload/52860/52860fig2.jpg" /> 图2:复制A和B.复制一个副本（左）被链接到细胞计数窗口并保持在50％放大倍率的一致性，从 ​​得分手，同时得分手副本B（右）可以放大到个人的偏好。 2.评分FP使用丹佛乳头协议二分法关键 <ol><li>对于每个候选乳头状拉，使用以下二分键（步骤2.2-2.5），详述的标准，以确定它是否是一个FP。参考图3进行目视被分类为FP乳头以及那些是在过程中的每个步骤拒绝。 <img alt="图3" src="/files/ftp_upload/52860/52860fig3.jpg" /> 图3:FP与拒绝乳头。图3a是与几个合格的FP舌头，而3B-3E已经盘旋违反规则的每一个领域。 （3b）的区域是无定形物质; （3C）乳头过小;比起那些周围3d）的乳头是蓝色的，（3E）乳头凹入相比，那些在其周围。 </li><li>形状<ol><li>确定候选人乳头是无定形（无形）。在50％的缩放查看是否有一个普遍公认的几何形状（椭圆形，立方形，圆形）。 </li><li>如果有一个几何形状（参见图3a），移动到步骤2.3。 </li><li>如果疗法e是没有几何形状（ 见图3B），进入细胞计数窗口，然后单击"类型1"在副本中的（50％缩放），点击候选人乳头，以将其标记为无定形，而不是一个FP。点击在这个和随后的步骤1-4型允许射手知道他们已经解决了所有的候选乳头和分类哪些规则被拒绝，这将在讨论有助于步骤2.7.2结构时侵犯。 </li></ol></li><li>颜色<ol><li>参阅复制，在50％的缩放，并确定是否有任何颜色区分在舌头的表面上。如果是这样，继续下一个2.3.2。如果不是，请不要使用颜色作为决定因素;移动到步骤2.4。 </li><li>确定该候选乳头比组织或乳头周围打火机（参照图3a）。如果有部分候选人乳头一直保持粉红或染色较浅，转到步骤2.4。 </li><li>如果考生乳头是蓝色及周边乳头打火机（ 见图3C），进入细胞计数窗口，然后单击"类型2"的细胞计数。在副本中，点击候选人标记为太蓝，而不是一个FP。 </li></ol></li><li>尺寸<ol><li>使用复制B，放大到距离其中一个可以舒适地看应聘者乳头的轮廓。 </li><li>点击在线工具和整个候选乳头的最长尺寸测量。点击"分析"，然后"措施。"如果测量长度为0.5毫米或更大（ 见图3A），转到步骤2.5。 </li><li>如果测得的长度为0.499毫米或更小，测量一次以确保准确性。如果仍然0.499毫米以下（请参见第F igure 3D），单击"类型3"的细胞计数窗口和副本的，点击候选人乳头，以将其标记为过小，而不是一个FP。 </li></ol></li><li>不景气<ol><li>使用副本的，如果评估候选人乳头要么是他制服飞行用舌头或升高的其余部分。如果乳头是在一个缝隙，而在该空隙，而不是舌表面的其它结构确定其高度。如果乳头低于周围的乳头较低（ 见图3E），用"4类"，并在副本中，点击候选乳头凹入的，而不是一个FP，以纪念它。 </li><li>如果候选乳头要么是均匀的高度与舌的其余部分或升高（参见图3a），用"类型5"点击它，并标记，这是一个FP。这种类型的共5个是原始FP得分。不要关闭该副本在这一点上是ImageJ的不保存在电池反得分也从1-5类型的彩色痕迹副本A. </li></ol></li><li>保存复制A <ol><li>在笔记本的原始分数FP写下来。 </li><li>在细胞计数窗口中，单击"导出图片。"除经实验室所需的命名系统。这将允许缔约方会议开幕在未来的y一个保留从1-5类型的彩色标记。它不会保存原始FP得分。 </li></ol></li><li>质量控制<ol><li>分数每张照片由两个单独的得分手，比较7。 </li><li>如果较高的FP原始分数是内的下部的FP原始分数的10％，对于一个共识的FP得分平均的两个分数在一起。如果两个FP原始分数相差超过10％，既拉得分照片在屏幕上。 <ol><li>使用计数器类型1-4，以帮助不符的讨论和赋予最终达成共识得分。如果没有共识在讨论中达成，采取从得分休息。在稍后一点，rescore的形象和讨论。如果一个共识仍然无法达成，拉在第三得分手。让他们得分的形象和对话与其他两个。 </li></ol></li></ol></li></ol> 3.培训进球使用DPP <ol><li>背景训练<ol><li>有两个得分手算上各种图像，单独得分并定期赋予，以确保它们是用相同的标准。只有当个别计数一致内彼此为每个图像的10％打进时是方法（以上定稿）认为是可接受的，并称为DPP。 </li><li>选择15的官方图片和15个训练图像（ 图补充2-17）顺序得分。如果图像中选择视为不可数，跳过图像，然后选择下一个连续的图像。 注意:这些图像在不同的质量和大小，因为他们选择，而实验室仍然掌握正确的摄影技术。其中一个选择的图像被视为不可数通过这个主计票过程，但留在确保得分手能够在需要的时候作出这样的电话。因此，也有给学员虽然只打进了15 16的图像图像。 </li><li>使用DPP，有上面提到的得分手算上15训练图像，并创建一个共识每个分数。 </li><li>给学员15的官方图片，并要求使用米勒和兰迪建立一个基线得分。记录分数。进行集体培训这一步跟随3.2。一旦步骤3.2完成后，有再次得分手得分15的官方图片，并记录他们的分数。 </li></ol></li><li>培训对民进党后续射手<ol><li>给训练得分手的第一训练图像和演示民进党对这一形象的几个乳头。讨论推理选择或拒绝每个乳头为FP。让学员继续练习得分为该照片的休息和与商谈的共识号（GOT的实验室的共识数量和类型1-5标记，见补充图1）。对于此照片讨论的任何差异无关的10％，以确保DPP的理解。 </li><li>随后给出得分第二训练图像，并让他们得分使用DPP。 </li><li>如果学员原始FP得分为十之内既定的共识％，从3.1.3节数，让学员进入下一个图像，然后重复步骤。 </li><li>如果分数超出百分之十的范围内，使用实习生的代表类型1-5了解和识别不一致，然后解决民进党的认识不符彩色标记。 </li><li>给予见习机会rescore图像，并验证新的得分一次。如果成功的话，见习移到下一个图像，直到所有图像都完成。 </li></ol></li></ol>

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<li>McNamara, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+Taste+summative+evaluation+report+prepared+for+Denver+Museum+of+Nature+%26+Science%5BTitle%5D)+AND+%22Independent+Evaluator%22%5BJournal%5D)">Genetics of Taste summative evaluation report prepared for Denver Museum of Nature & Science.</a> Independent Evaluator. , Chicago IL. (2012).</li>
<li>Arvidson, K., Friberg, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+taste%3A+Response+and+taste+bud+number+in+fungiform+papillae%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Human taste: Response and taste bud number in fungiform papillae.</a> Science. 209 (4458), 807-808 (1980).</li>
</ol>

作者宣称，他们有没有竞争的财务权益。

使用DPP，在图像的方差计算跨越独立的得分手，并在记分员的数量显著下降。虽然这种方法使视觉FP密度分析，无电视显微镜少主观的，但应注意的是，这种方法只分数的FP。民进党不能保证归类为FP结构有味道的毛孔，因此味觉乳头，也不该计数是反射型实味蕾密度20,23这种方法的本质.The是，它通过一致性和精度确立的可靠性;然而，它可以被偏压的结果一致的方向。要通过更严格FP特征的形态，创造一个二分法键使用味蕾密度（ 例如 ，电视显微镜）的真实措施将超出这一study.However的范围测试相对于其他方法的精度，这种方法解决了米勒＆里迪的负责人向现场的FP更表征需要，以便对它们的形态和定量2共识完成。此外，他们表示，这可能表征"澄清一些学科之间的差异，并解释了一些不同的调查结果当中2"通过提供定量的统一的方法，在众多的FP的味道和与营养的作用不符相关研究可以得到纠正。 二分法键可以为不同的图像捕捉技术进行调整。为测量尺的已知距离可以很容易地设置在每个实验室和经济衰退，如果用保鲜膜或玻璃被放置在舌头的图像捕捉可以打折的直径。在GOT实验室，我们发现中性舌提供乳头的最佳定义。这可能是由于光在实验室造成保鲜膜或玻璃光反射回进入相机创建的眩光，使得驴量sment困难。一些实验室已经表示，他们发现染料更具挑战性来算，但得到了实验室发现了一个蓝色的稀释食用染料在1:36设于广大的舌头菌状和丝状乳头之间的理想反差的浓度。 二分法键FP表征的建立将允许研究人员在不同的实验室，采用多种图像采集方法，始终如一地分析鞋舌上的结构和有信心，其结果的报告。最后，使用二分法关键可能有目的的科学方法论的其他领域的结构或物体的性质所提供的研究，以研究数字不一致。

乳头在舌头表面的可见的颠簸。有四种类型的乳头:菌状（FP），叶状，circumvallate和丝状。而丝状乳头分布在舌头的表面，circumvallate仅在舌头的后部局部，和叶状上发现的两侧。与此相反，FP位于朝向舌的前部。 FP，叶状和轮廓乳头被认为是味觉，因为他们含有味蕾，而丝状没有潜力。味蕾簇负责检测化学促味剂和转导的刺激成在大脑味觉信号的细胞。唾液和食物分子通孔称为味孔，其中味道分子然后在激活味觉细胞的潜在进入味蕾。 有趣的是，也有味蕾密度较大的个体差异，在舌头的组织学研究首次发现从18具尸体（米勒，1988年）1。味蕾本身是不是在舌头表面可见的，但是，味孔是可见的使用而导致米勒和兰迪（1990）适当的放大倍率，照明和染色技术发展的方法来识别利用电视显微镜生活品味毛孔，菌状乳头时，人类2。一些实验室已经修改了他们原来的视频显微技术，采用静态摄影，很少或没有放大。然而，由于菌状乳头更容易形象化比是使用这些后者技术毛孔，实验室转向代替味孔的计数菌状乳头。在这样做时，假设菌状乳头密度是一个合理的代理度量味道孔密度，尽管许多菌状乳头不含有味道孔或味蕾3;不仅FP更加明显，比circumvallate和叶状乳头访问，但它们的图像捕捉少TI我比消耗通过视频显微2,4的 ​​替代方法，味道孔隙分析。在1990年，米勒＆芦苇丛用这种技术，发现味道孔隙密度的值与FP密度3，将其进一步证实在Bartoshuk 等 5除此解剖关系，米勒＆芦苇丛还发现，在上的8个人一半的味道孔隙密度分布也被评为蔗糖，氯化钠，丙硫氧嘧啶（PROP）比没有其他8个人3作为显著更加激烈。通过Bartoshuk 等人后来的工作。（1994）观察到的阈上PROP味道强度和菌状乳头密度之间的相关性，以及味道的孔密度，在42受试者5。这种便捷的方法导致了有影响力的研究报告说，其主体舌头有很多乳头引起更强的反应，许多促味，其中包括促味剂苦苯硫脲（PTC）和PROP 6,7。 PTC和PROP的精心研究促味经常使用的，由于一个人的品味敏感性表型和基因型他们之间的明确联系。人口研究表明，人谁具有基因，TAS2R38的纯合隐性diplotype，有一个显著较低灵敏度PROP比基因8-10的显性或杂变体的携带者。这种遗传现象最早是在1932年时报道亚瑟L.福克 ​​斯宣布了他的发现"味盲"到PTC 11。人与人之间能品尝这些苦味化合物，品味的报道力度可以改变到难以忍受的痛苦的6微微从任何地方不快。为了解释方差不能由TAS2R38进行说明，该理论被提出，它可以归因于FP对舌4的密度。 由于口味的研究进展，FIELD组分为围绕这个理论两个学派关于FP和苦味敏感性的作用。虽然许多研究证实了原来的主张FP密度在PROP的一个因素敏感性4,6,12,13，出现了一个小的，但很重要的应急已发现的证据来反驳，报告一个无法复制的作用10， 14,15,16。 Delwiche 等 。 （2001）警告说，趋势是高度可变的; FP密度不均匀帐户中跨学科苦味灵敏度的差异，并且是只证明的，用于与至少一个中等灵敏度受试者PROP 7。除了 ​​使用上如何量化FP不太严格定义的米勒＆芦苇丛方法，值得注意的是，与异常的比弗达姆后代研究15由Fischer进行的。 等 ，多数上述研究了小样本是重要大小这也可能有助于对不一致的结果FP的味道的作用。 简单地说，米勒和兰迪的开创性方法纸（1990），作者舌头染成蓝色的量化FP因为FP保持粉红色，而丝状乳头吸收染料变成蓝色。他们归类的FP为"圆形粉红结构约0.5毫米的直径"2。虽然作者建议，使用此方法作进一步的分析工作之前，FP的特性应更严格的定义，并得出结论，该变异在文献中已经指出，"可能是由于不同的受试者群体，不同的方法以及不同的调查"2，其研究导致了普遍接受的特性的FP是圆17,18，大2，粉红色或染色较浅2，和升高4。从表面上看，这些特性显得非常直截了当。在口味实验室的遗传学（GOT实验室或实验室），使用的会晤HOD在排位复杂化了记分个人企图和优先的上述特性的一些时的重要性，但不是全部，所述特性是本。这些并发症包括小乳头的出现粉红色的更大的乳头中间;整个舌吸收蓝色染料从而消除基于颜色变化分类;长圆形乳头，而不是圆形;和不具有变化高度，所有这些都有助于得分报告相同图像的广泛多样的FP计数舌片。 这些变化在分析使我们回顾文献，以查明主观性，识别任何异常普遍接受FP特性，并最终制定一个协议，以准确，客观地界定和得分FP。 首先，FP形状在文献中描述为圆形，蘑菇状结构17,18。然而，除了显着离子也被提及。米勒强调，"菌状"一词是用词不当，而且许多FP不是蘑菇形，但可以广泛地变化在大小和形态18。梅利斯等 。 （2013年）也描述了几个乳头那些被认为"歪曲"，其中FP直径在一个方向上比在另一个方向19长至少两个标准偏差。此外，郑和罗宾逊发现表明FP通过染色时，它们介于平顶，以细长的外观20。注意到上述例外情况，一个FP的特征是圆形和蘑菇状出现过于狭隘的定义。 其次，FP色染色蓝染舌后描述为7"，背景为蓝色粉红色圆圈"。虽然染色较浅是计划生育的最一致的标准，在整个文学它仍然没有一个统一的预选赛。郑和<html> ;罗宾逊指出，FP并非总是淡染，使识别困难和不确定20。看来那么不同的方言吸收染料而对比度很多舌头明显，有的成为完全的蓝色，没有区别，而其他人立刻失去了蓝色染料21的任何痕迹。 第三个特点，FP大小，是其中纸张相当一致的，范围从0.5mm到0.97毫米6,18。在文献米勒指出，有乳头在舌头上的背侧前两种尺寸范围。具有较大直径的乳头主要是菌状和圆锥形乳头，而具有较小直径的乳头大多丝状18。然而，唯一的例外是比弗达姆后代研究21，尺寸并未以确定是否一个结构是在文献中的FP，但被提供为已划分为"菌状"乳头。 _content"&gt;最后，海拔是使用此标准靠近舌的边缘时列为第四FP特性4虽然米勒指出丝状和菌状乳头之间的区别是困难的。他给FP的两个例子，在标高变化很大，一个是0.8mm的高度，而另一个是小于0.1毫米的高度，但仍然有两个味道毛孔清楚地观察到18。这一观察也随访和由Shahbake 等人证实。4 这些定义和报告异常显示，味道领域早已认识的不一致性在表征FP与常用的协议从米勒＆芦苇丛的缺点。事实上，米勒和兰迪告诫领域，以进一步确定FP 2的功能。我们假设得分手被的特点给予不同的权重，当爸的重要性不同优先illae未能满足每标准，例如，当一个乳头很大并且蘑菇形，但染成蓝色。据推测，先前发表的研究调查可能确实，当与小样本大小结合，可能占不同的结果，从使用这种方法的到来一样。 该实验室得到了通过制定一项准则，用客观的测量定义每个特点和优先的特点，指出哪些可以优先处理的分析中FP方法这一空白。这种方法是丹佛乳头协议（DPP），与FP明确和鲜明的特色二分项，以确保从舌的数码照片精确和可重复计数。民进党是一个建议的方法，以规范以前使用的米勒和兰迪的方法，从而消除个别解释，并确保更一致的结果进行分析时FP密度。通过这样做，民进党可以用来更自信ðetermine FP的味道的作用。 所采取的受试者的舌图像是由公民科学家的实验室（得分）谁是本研究的真实对象进行分析。该实验室的核心公民科学家由来自社会各界不等会员年龄（岁）从16到80年代中期。由博物馆在2012年进行的一项总结性评价报告描述公民科学家为具有科学有浓厚的兴趣，并与三分之二已经获得了一门学科22大学学历。候选公民科学家通过口耳相传目前招募的，必须成功完成的采访过程在博物馆老将志愿者，并接受在博物馆的永久生创展，远征健康，试用期，他们有机会申请在一个位置之前实验室。一旦接受进入实验室，公民科学家经过12周的培训，成为认证的注册人的主体秒。在此之后的认证，他们能够参加培训模块，在实验室各种技术（ 如乳头计数，DNA提取）和下列额外的认证和定期的质量控制措施，这些公民科学家有机会积极参与数据分析。公民科学家志愿者他们的时间和他们的博物馆贡献没有得到补偿。

<table><tbody><tr><td>Image J</td><td>National Institutes of Health</td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij&quot;&gt;http://rsb.info.nih.gov/ij&lt;/a&gt;</td><td></td></tr><tr><td>Food dye, Deep Blue Shade</td><td>Esco</td><td>http://escofoods.com/blue-food-coloring.html</td><td></td></tr><tr><td>Sterile Rayon Tipped OB/GYN Applicators</td><td>Puritan</td><td>25-808 1PR</td><td></td></tr><tr><td>Grade 1 Filter Paper</td><td>Whatman</td><td>Whatman No.: 1001-325</td><td></td></tr><tr><td>Coolpix P100 (10.3 megapixels) and P500 (12.1 megapixels)</td><td>Nikon</td><td>http://imaging.nikon.com/lineup/coolpix/</td><td>neither model appears to be available on the website any longer</td></tr><tr><td>Paper Hand Towels</td><td>Kleenex</td><td>https://www.kleenex.com/HandTowelsDetail.aspx</td><td></td></tr></tbody></table>

denver papillae protocol for objective analysis of fungiform papillae

丹佛乳头议定书的目标是用一个两分的关键定义和优先菌状乳头（FP）的特性，以确保得分手之间稳定的得分。这个协议建立关的需要已经出现了从过去的二十年使用的FP作为味道孔密度的代理味道研究这一点。 FP密度历来被米勒用芦苇丛及1990年的刻画它们的形态分析的:圆形，染色较浅，大，和高架。在这项工作中，作者事先警告的需要FP形态严格的定义进行概述。尽管这样的行动呼吁，跟进文学一直稀少，多数研究继续举米勒和兰迪的原创作品。因此，FP密度报告已经充满变数，并结合样本量小，可以在FP的味道敏感性的作用有助于不符的结论。味觉实验室的遗传学探索了这种明显的INCON一致性在数量，发现得分被单独优先化这些特征的重要性有所不同，并具有用于当一个乳头有一些没有指导，但并非全部的FP的报告品质。这种主观性的结果是一样的舌象的高度可变的FP计数。丹佛乳头协议已经制定通过使用二分法关键是进一步明确和优先的提出米勒和兰迪特点的重要性，以解决这个问题的后果。该方法可以帮助建立量化FP研究人员在口感和营养研究领域的标准方法。

下面是使用基于圆形，大，粉红色的领域标准的常用米勒和兰迪方法论FP鉴定时打进了代表照片由两个单独的得分手或染色较浅，和高架，然后是同样的照片打进两个得分手使用DPP。显示的计数是代表用原来的方法相比，使用DPP低方差观察到的高方差。 <img alt="图4" src="/files/ftp_upload/52860/52860fig4.jpg" /> 图4:米勒＆芦苇丛定量VS DPP定量左边的图片代表使用米勒＆芦苇丛方法二记分计数。右边的图像代表使用DPP 2得分手&#39;罪名。红色是得分手1，黄色是射手2，绿色是得分手1＆2。 DP的统计可靠性P含量采用混合线性模型来评估可变性10先前研究。简单地说，生成数据，以评估的作用民进党分数一致性扮演，十五图像梅开二度。在第一批分数，民进党天真得分手使用的米勒和兰迪方法2。记分然后训练使用的DPP和图像被单独rescored而不在第2.7节详细描述的质量控制（ 图5）中描述的最终共识步骤。然后在混合模型中使用的FP的得分数据组以评估由于协议（ 图6），内射手变性和这两者之间的相互作用将合并的差异。这个模型显示用相比DPP（p值&lt;1×10 -6）与DPP导致较高的计数由6.99（SE = 0.99）的米勒＆芦苇丛方法时在得分一个显著差异;在得分的可变性的5.2％是由于训练，25.9％由于射手，其余因可变性特定于图像10。接着，以产生数据，以确定内射手变性，一系列随机顺序30的图像进行评分由11个人。不知情的得分手，这一系列的图像包含重复的三幅图像。我们观察到比较明显的图像得分变化剧烈的区别versusrepeated图像。 <img alt="图5" src="/files/ftp_upload/52860/52860fig5.jpg" /> 图5:DPP-朴素VS邮政民进党培训面板提供了评审分数为15幅箱图前后的丹佛乳头协议培训。对于每一个箱线图，一个填充圆圈表示平均，而一个开放的圆圈表示离群值。 <img alt="图6" src="/files/ftp_upload/52860/52860fig6.jpg" /> 图6:变化射手差异的灰色代表叔他方差前民进党培训个人的得分手。黑色是一样的得分手后民进党培训。

Watch this Scientific Journal Video about Denver Papillae Protocol for Objective Analysis of Fungiform Papillae at JoVE.com

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

在这里，我们提出了一个协议，以测量从数码照片菌状乳头密度。此方法构建的优先次序和客观的特征指标到由米勒和兰迪（1990年）菌状乳头原来的工作描述。

为菌状乳头的客观分析丹佛乳头协议

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

denver-papillae-protocol-for-objective-analysis-of-fungiform-papillae

Research

JoVE Journal

Neuroscience

14.3K 次观看.  Denver Museum of Nature & Science. 在这里，我们提出了一个协议，以测量从数码照片菌状乳头密度。此方法构建的优先次序和客观的特征指标到由米勒和兰迪（1990年）菌状乳头原来的工作描述。 

视频: Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

科研

医学

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

神经科学

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

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 ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

味觉考试: 一个简短的验证测试

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. <a href="https://www.jove.com/video/62300" target="_blank">Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells</a>. J. Vis. Exp. (2021)
In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

Isolation of Circumvallate Papillae CVP Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

从小鼠模型中分离环状（CVP） 上皮:从小鼠舌中分离 CVP 上皮的酶促程序

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)
In this video, ...

为菌状乳头的客观分析丹佛乳头协议

摘要

探索更多视频

技术收集人舌菌状（味道）乳头

人力蕈状味觉乳头细胞的分离和培养

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

协议星形胶质细胞的三维共聚焦形态分析

口腔生物膜取样在健康儿童中的微生物分析

味觉考试: 一个简短的验证测试

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序

为菌状乳头的客观分析丹佛乳头协议

摘要

探索更多视频

技术收集人舌菌状（味道）乳头

人力蕈状味觉​​乳头细胞的分离和培养

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

协议星形胶质细胞的三维共聚焦形态分析

口腔生物膜取样在健康儿童中的微生物分析

味觉考试: 一个简短的验证测试

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

从小鼠模型中分离环状（CVP） 上皮:从小鼠舌中分离 CVP 上皮的酶促程序

人力蕈状味觉乳头细胞的分离和培养

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序