蒂姆·戴利和安德鲁·史密斯在提供早期的许多有用的计算讨论;蒂姆·戴利写了协议5.计算资源是由高性能计算集群在南加州大学提供了必要的程序rotate_translate_cylindrical。这项工作是由美国国立卫生研究院授予＃GM098536（MDD）的支持。

所有的过程和人员由南加州学院的大学动物护理和使用委员会（IACUC），协议＃11394批准。 1.解剖短肛和准备<ol><li>通过二氧化碳过度曝光安乐死一个性成熟的雄性小鼠，根据相关机构的动物护理和使用委员会（IACUC）提出的协议。 </li><li>躺在仰卧位的动物，并通过施加压力拖延阴茎大拇指外侧包皮开幕。 </li><li>一旦阴茎旷日持久，通过包皮尽可能延长组织。 </li><li>用剪刀，剪断阴茎体近端，其中阴茎骨所在的龟头。 </li><li>转移解剖阴茎1.7毫升管中，加入200μl自来水。使某些阴茎在液体完全浸没。 </li><li>孵育在水中的组织在约50℃下3-5天。 </li><li&gt;适当温育后，从阴茎骨除去周围组织，用解剖显微镜下用镊子。轻轻喷70％乙醇推下剩余的组织和清理骨头。 </li><li>放置解剖阴茎骨与盖打开一个新的离心管中。请假封顶开O / N为干骨。 </li></ol> 2.显微扫描<ol><li>按一个显微扫描圆柱支架插入花店泡沫砖创造花店泡沫的圆柱体。 </li><li>提取花店泡沫缸和切割片〜2-5厘米厚。 </li><li>干燥后推入的bacula花店泡沫，围绕单个片的外围扫描过程中，尽量减少干扰。骨骼的精确定位应该说允许在协议4个人标本的正确识别。 </li><li>轻轻将切片与嵌入式骨头进入显微持有人。 </li><li>获得显微扫描。在小鼠的bacula 14的情况下</sup90 kVp的，155μA为0.5mm铝过滤器，每180（360覆盖）750预测，曝光时间:&gt;，我们根据以下设置在南加州大学分子影像中心使用的uCT50扫描器（SCANCO医疗AG，Bruttisellen，瑞士） 500毫秒，为15.5mm的体素尺寸。 </li></ol> 3.显微加工:转换一个.DCM堆栈到单个名为.xyz文件注意:每个显微扫描产生一个堆栈.DCM的，或"DICOM"，即表示通过对象拍摄图像片段文件。所有下游计算几何需要平面名为.xyz文件，这是一个简单的包含四列的文本文件 - 在x，y和每个像素的z坐标的，并且所述像素的强度，范围从-5000（黑色）到5000 （白色）。超过3000像素的阈值通常工作以及定义骨骼的阈值。 <ol><li>安装Python（www.python.org）和Python模块命令，DICOM，PYLAB，SYS和numpy的。 </li><li>打开"01_process_dicom.py"{} Figshare用任何文本编辑器，在变量部分，改变路径，像素阈值和目录名是必要的。 </li><li>运行"蟒蛇01_process_dicom.py"。进展情况将被打印到屏幕上。在每个步骤3.2命名的目录，新文件由命名;例如，directory_name.PT3000.xyz，其中PT3000指示像素阈在步骤3.2表示。 </li></ol> 4.显微加工:细分出单独的标本名为.xyz文件<ol><li>与库RGL安装R（https://www.r-project.org/）。 </li><li>用任何文本编辑器打开文件02_segment_dicoms.r&#39;{} Figshare。根据变量部分，更改路径名称指向上述协议3中创建的名为.xyz文件。 </li><li>从内部R，运行命令"源（&#39;02_segment_dicoms.r&#39;）"（不包括双引号）。 </li><li>在协议3中创建的名为.xyz文件的三维图像出现后，输入numbe在整体名为.xyz文件标本河然后贴上标签并选择使用滚动和缩放功能，每个样本点。 注:在后台，独立名为.xyz文件将每个样品上进行。这些出现在一个新的目录，例如，XYZ_FILES_PT3000，其中PT3000表示使用的像素阈值。 </li></ol> 5."对齐"标本名为.xyz文件到通用坐标。 <ol><li>打开Python脚本"03_transform.py"{} Figshare，这需要额外的模块mattdean_modules.py {} Figshare，以及两个自立的应用程序:"rotate_translate_cylindrical"（https://github.com/timydaley/dean_cylindrical_tranform）和"qconvex"（www.qhull.org/html/qconvex.htm）受此脚本中使用。 </li><li>根据变量部分，确定全路径名mattdean_modules.py，rotate_path和qconvex_dir。此外，确定到包含个体.xy目录的完整路径步骤4中创建ž文件。 </li><li>运行03_transform.py，它创建每个试样的新文件的后缀.TRANSFORMED.xyz。 </li></ol> 6."切片"不结盟标本名为.xyz文件识别半地标。 <ol><li>打开并运行Python脚本"04_identify_landmarks.py"{} Figshare。在变量部分，确定全路径名包含.TRANSFORMED.xyz文件的目录。此脚本标识可以用来量化该结构的尺寸和形状802半标。 </li></ol>

<ol>
	<li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrics+morphometrics.">Geometrics morphometrics.</a> Annu. Rev. Anthropol. 36, 261-281 (2007).</li><li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. 6, (2005).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2012).</li><li>Bookstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Morphometric tools for landmark data: geometry and biology. , (1991).</li><li>Rohlf, F. J., Marcus, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Revolution+in+Morphometrics.">A Revolution in Morphometrics.</a> Trends. Ecol. Evol. 8 (4), 129-132 (1993).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D., Fink, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2004).</li><li>Rohlf, F. J., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensions+of+the+Procrustes+method+for+the+optimal+superimposition+of+landmarks.">Extensions of the Procrustes method for the optimal superimposition of landmarks.</a> Syst. Zool. 39 (1), 40-59 (1990).</li><li>Gunz, P., Mitteroecker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semilandmarks:+a+method+for+quantifying+curves+and+surfaces.">Semilandmarks: a method for quantifying curves and surfaces.</a> Hystrix. 24 (1), 103-109 (2013).</li><li>Gunz, P., Ramsier, M., Kuhrig, M., Hublin, J. J., Spoor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+bony+labyrinth+reconsidered,+introducing+a+comprehensive+geometric+morphometric+approach.">The mammalian bony labyrinth reconsidered, introducing a comprehensive geometric morphometric approach.</a> J. Anat. 220 (6), 529-543 (2012).</li><li>Mitteroecker, P., Gunz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+geometric+morphometrics.">Advances in geometric morphometrics.</a> Evol. Biol. 36 (2), 235-247 (2009).</li><li>Bookstein, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landmark+methods+for+forms+without+landmarks:+morphometrics+of+group+differences+in+outline+shape.">Landmark methods for forms without landmarks: morphometrics of group differences in outline shape.</a> Med. Im. Anal. 1 (3), 225-243 (1997).</li><li>Gunz, P., Mitteroecker, P., Bookstein, F., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. , 73-98 (2005).</li><li>Oxnard, C., O'Higgins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+Clearly+Needs+Morphometrics.+Does+Morphometrics+Need+Biology?.">Biology Clearly Needs Morphometrics. Does Morphometrics Need Biology?.</a> Biological Theory. 4 (1), 84-97 (2009).</li><li>Schultz, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+baculum+size+and+shape+variation+in+mice.">The genetic basis of baculum size and shape variation in mice.</a> G3. 6 (5), 1141-1151 (2016).</li><li>Schultz, N. G., Lough-Stevens, M., Abreu, E., Orr, T. J., Dean, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculum+was+gained+and+lost+multiple+times+during+mammalian+evolution.">The baculum was gained and lost multiple times during mammalian evolution.</a> Integr Comp Biol. 56 (4), 644-656 (2016).</li><li>Dines, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+selection+targets+cetacean+pelvic+bones.">Sexual selection targets cetacean pelvic bones.</a> Evolution. 68 (11), 3296-3306 (2014).</li><li>Adams, D. C., Ot&#225;rola-Castillo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=geomorph:+an+R+package+for+the+collection+and+analysis+of+geometric+morphometric+shape+data.">geomorph: an R package for the collection and analysis of geometric morphometric shape data.</a> Methods Ecol. Evol. 4 (4), 393-399 (2013).</li></ol>

The authors declare that they have no competing financial interests.

在上述协议中的关键步骤是:1）解剖的bacula，2）收集显微图像，3）显微输出转换到XYZ的平面文件坐标，4）分割出各试样的点云，5）将每个试样标准化的坐标系，和6）限定的半标。这些步骤很容易被修改，以适应不同的对象。 这些方法可能可以被应用到实质上是"棒状"，或者至少不太弯曲的任何对象。对象曲线回自己成为"U形"不能被目前分析，因为切片（ 图1G）...

形态测量的领域包括的方法来量化的大小和生物形式的形状，在科学调查1，2，3，4，5，6的一个基本步骤的分集。传统上，大小和形状的统计分析开始于识别上的生物结构的地标，然后测量线性距离，角度和比例，这可以在一个多变量的框架进行分析。里程碑式的基于几何形态特征是保留的地标空间位置，通过分析和可视化5保存从数据收集几何信息的方法。广义普鲁克分析（GPA）可以应用到消除位置，规模，和地标旋转变化，以产生是m标本之间的对准inimizes他们的平方差-剩下的就是形状相异7。 任何形态分析的一个重要概念是同源性，或一个可以可靠地识别代表该标本或结构之间对应生物学意义的和离散的特征的地标的想法。例如，人头骨有同源流程，椎间孔，缝合线，和管道，可以使形态学分析。不幸的是，对应的地标标识是在许多生物结构困难，特别是那些具有光滑表面或曲线8，9，10。 我们下面的办法使用计算几何这个问题。的一般工作流程，以产生可以被表示为点的云的物体的三维扫描，然后旋转和转换，该转换点云，使得所有specimens被定向一个共同的坐标系上。然后我们数学定义从物体的特定区域的半标。放在这些地区离散半地标生物任意11。开展GPA和随后的统计分析可以产生不良的文物8，12，因为随意放置地标可能不是生物同源。因此，我们允许这些半地标数学"滑动"。此过程减少结构之间的电势差。如别处认为这里使用的滑动算法是适当的量化缺乏类似解剖区域容易识别相应的地标3，6，8，10，11，12。这些方法各有其利mitations 13，但应该适应不同尺寸和形状的物体。 这里，我们说明这个方法是如何在近一个鼠标阴茎骨14的研究应用，在已经获得和哺乳动物进化15期间丢失多个独立倍阴茎骨。我们讨论解剖和制备的特定骨的，所述阴茎骨（协议1），生成显微图像（协议2），并且这些图像以一种格式，使所有下游计算几何变换（协议3和4）。完成这些步骤后，由〜100K XYZ坐标每个试样表示。然后，我们通过一系列的变革，有效地调整所有标本到一个共同的方向（协议5）走，然后定义从对准标本半地标（协议6）。协议1-4应该是相似的，无论被分析的对象。协议5和6号议定书是SPEcifically专为阴茎骨，但我们希望通过详细介绍这些步骤，研究者可以想像的修改，这将是对有关他们感兴趣的对象。例如，这些方法改进了用于研究鲸骨盆和肋骨16。

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting scissors</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">470106-338</td>
			<td style="width:167px;">Most sizes should work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting Forceps, Fine Tip, Curved</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">82027-406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">1.7 mL microcentrifuge tube</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">87003-294</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Absolute Ethanol</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:79px;">CAS 64-17-5</td>
			<td>To be diluted to 70% for dissections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Floral Foam</td>
			<td style="width:287px;">Wholesale Floral</td>
			<td style="width:79px;">6002-48-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">uCT50 scanner&#160;</td>
			<td colspan="2">Scanco Medical AG, Bruttisellen, Switzerland</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection, microct scanning and morphometric analyses of the baculum

现代形态测量提供了强大的方法来量化的大小和形状的变化。一个基本要求是，确定地标坐标列表;然而，这样的坐标必须代表整个标本同源结构。虽然许多生物对象包括容易识别标志，以满足同源性的假设，许多缺乏这种结构。一个可能的解决方案是在数学上横跨试样代表相同形态区域中的对象在地方半标。在这里，我们说明了一个新近开发的管道数学定义从鼠标阴茎骨（阴茎骨）半地标。我们的方法应该适用于广泛范围的对象。

在协议6中产生的半地标XYZ坐标可以直接导入到任何基于具有里程碑意义的几何形态测量分析17。计算管道上面已经用于研究小鼠的bacula 14，以及鲸骨盆和肋骨16。上半的地标计算定义的更多细节，这里提出，以试图帮助研究人员可视可能被修改，以适应他们感兴趣的特定对象的步骤。该阴茎骨包含了被利用来自动化某?...

Watch this Scientific Journal Video about Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum at JoVE.com

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

许多生物结构缺乏容易定义的地标，从而难以应用现代形态的方法。在这里，我们举例说明的方法来研究的小鼠阴茎骨（在阴茎骨），包括夹层和显微扫描，随后计算方法来定义用于量化的尺寸和形状的变化的半标。

在中短肛解剖，显微扫描和分析形态

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

dissection-microct-scanning-and-morphometric-analyses-of-the-baculum

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.5K 次观看.  University of Southern California (USC). 许多生物结构缺乏容易定义的地标，从而难以应用现代形态的方法。在这里，我们举例说明的方法来研究的小鼠阴茎骨（在阴茎骨），包括夹层和显微扫描，随后计算方法来定义用于量化的尺寸和形状的变化的半标。 

视频: Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

科研

生物工程

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

视频率扫描共聚焦显微镜和内窥镜

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

用于玻璃化和变暖步骤中囊胚行为客观评估的形态学协议

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

在中短肛解剖，显微扫描和分析形态

摘要

探索更多视频

此视频中的章节

刀口扫描显微镜的样品制备，图像和分析协议

视频率扫描共聚焦显微镜和内窥镜

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

阿普利西亚单一神经元电生理学和分子分析的甘利亚制备

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

用于玻璃化和变暖步骤中囊胚行为客观评估的形态学协议

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

在中短肛解剖，显微扫描和分析形态

摘要

探索更多视频

此视频中的章节

刀口扫描显微镜的样品制备，图像和分析协议

视频率扫描共聚焦显微镜和内窥镜

膀胱膀胱分析各种啮齿类动物模型中使用的增广的生物材料评价

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

用于玻璃化和变暖步骤中囊胚行为客观评估的形态学协议

通过叶反射和叶绿素荧光分析同步测量的光合行为评价

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

阿普利西亚单一神经元电生理学和分子分析的甘利亚制备