This work was supported through an Innovative Engineering for Health award by the Wellcome Trust [WT101957], the Engineering and Physical Sciences Research Council (EPSRC) [NS/A000027/1], and a National Institute for Health Research Biomedical Research Centre UCLH/UCL High Impact Initiative. Jan Deprest is being funded by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO; JD as clinical researcher 1.8.012.07). Danail Stoyanov receives funding from the EPSRC (EP/N013220/1, EP/N022750/1), the EU-FP7 project CASCADE (FP7-ICT-2913-601021), and the EU-Horizon2020 project EndoVESPA (H2020-ICT- 2015-688592). Sebastien Ourselin receives funding from the EPSRC (EP/H046410/1, EP/J020990/1, EP/K005278) and the MRC (MR/J01107X/1). Marcel Tella is supported by the EPSRC-funded UCL Centre for Doctoral Training in Medical Imaging (EP/L016478/1).

1.目标制造<ol><li>喷砂<ol><li>制备316不锈钢板的厚度1.2毫米。用铅笔或指甲，画有40毫米×40mm见方到纸张上用尺子的帮助。 </li><li>剪下使用手动金属切割机的拉方。警告！观看手指。 </li><li>使用文件圆试样的角部和侧面。警告！他们是非常尖锐的;小心。 </li><li>制备的直木制或金属板的尺寸比不锈钢板稍大。将它切薄片;为了避免喷砂期间弯曲样品做到这一点。 </li><li>将装配在内部爆炸室。记得使用除尘器，并加以密封，内部爆炸室;否则，将砂子过程中星罗棋布。佩戴安全护目镜以保护眼睛。 </li><li>一个喷枪垂直位置和至少4-5厘米从金属表面。应用对照足OL的喷砂。戴上一块用木头副（1-2厘米厚）的样品，由于高压流砂可以变形的样本。在喷砂除锈，由一块木板的边缘或通过其他副紧紧握住的样本。 </li><li>重复在另一侧的喷砂如果它是希望具有两侧刻一个校正图案。 </li></ol></li><li>激光制图<ol><li>设计不对称圆圈的图案， 如图1。 </li><li>准备设计或者使用CAD软件或其他合适的编程语言的一个图画交换格式（DXF）文件。 注意:为方便起见，可以生成DXF文件在本文中提到的设计Python应用程序被提供为紧凑GUI应用程序16的一部分。 </li><li>导入DXF文件到激光切割软件。 </li><li>设置为背景蚀刻以下参数。激光功率:40％，扫描速度:80厘米/秒，频率:4000赫兹，传球次数:1。 </li><li>设置以下参数用于蚀刻图案。激光功率:40％，扫描速度2.1厘米/秒，频率:4000赫兹，传球次数:1。 </li><li>把工作平台上的样品，并使用该软件对准切割图案。 </li><li>激光进行切割后，用酒精浸泡它清洁样品。不要使用任何纸巾，因为它们通常离开不当残余物。 </li></ol></li><li>消毒<ol><li>包裹灭菌试样中的灭菌包，并在杀菌部（高压釜）插入。 </li><li>加水（不能蒸馏水），以高压釜，并按照用户指南/制造商的建议进行消毒的目标。 </li></ol></li></ol> 2.围手术期校准<ol><li>校准软件<ol><li>安装"endocal"内窥镜校准软件提供了GitHub上16包（遵循README文件中的指导文献）。 注:此软件包装OpenCV的摄像机标定模块17在一个易于使用的应用程序方便。所提供的应用程序运行在两种模式:在线和离线。在线模式直接从兼容的图像采集硬件获取的视频流。脱机模式允许装载型内窥镜的图像无论是从一个视频文件或与一些保存为图像文件的视频帧的一个文件夹。见自述文件支持的硬件和如何使用这两种模式的详细说明。 </li></ol></li><li>内镜视频采集 注:以下说明适用于在线校准（如上所述），但它们也适用于离线校准。 <ol><li>放置校准目标在无菌流体容器，如一个药罐。 </li><li>填与目标流体或类似的灭菌物质容器。 注意:例如，在胎儿镜PROCE的既定程序，目标流体是羊水。由于羊水的光学性质类似于食盐水18，19，可以使用无菌盐水的水，用于校准的胎儿镜。 </li><li>根据需要调整内窥镜的变焦和清晰度。 </li><li>浸入的内窥镜的流体，并在从类似于从该内窥镜稍后在所使用的解剖结构的距离校准目标的距离保持它。 </li><li>启动校准应用程序并启动摄像头采集。 </li><li>轻轻移动内窥镜的顶端为不同的看法，同时保持整个校准模式鉴于相机。为了获得最佳性能，保持围绕内窥镜的圆形视图中的校正图案的椭圆形的图例。 注:视频是可用于校准帧由一个虚拟图案覆盖所指出的，如在图3看到的。 </li><li>阿奎重的至少校准所需（如在endocal窗口指示）内窥镜摄像机视图的最小数目。 注:endocal当前版本的需要校准至少10内窥镜摄像机视图，以启发式选择的若干意见，其中校准误差显得微乎其微，并按照一个稳定的格局20。 </li><li>按下校准键，endocal窗口上所指示的，使用迄今所获得的图像，以启动校准过程。 </li></ol></li><li>保存和使用校准参数<ol><li>按指示的校准键保存在YAML产生的校准参数（"YAML是不是标记语言"）文件21。 </li><li>组校准参数插入相机矩阵和失真系数，正如OpenCV的摄像机标定模块17中说明。 注意:在执行校准之后，校准应用自动显示失真校正图象到原始内窥镜图像的右侧。 </li><li>纯粹的可视化和实时胎盘拼接11胎儿镜手术过程中使用失真校正视频饲料。 </li></ol></li></ol>

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The authors have nothing to disclose.

喷砂是在制造过程中的一个重要步骤，因为原料的金属表面突出地反映了内窥镜光，使得不可能对要检测的社交圈。是困难甚至用肉眼区分界（参见图5）。注意，示出的目标的表面已经用激光蚀刻。然而，这并没有减少光线的反射。 <img alt="图5" src="/files/ftp_upload/55298/55298fig5.jpg" /> 图5:不带喷砂校准目标应用。如从左侧的内窥镜视图看出，从材料表面上的内窥镜光眩光使得难以甚至对于肉眼区分界（有正好到大反射东南一个圆）。请注意，此靶的表面（ 即，"背景"）已经被蚀刻，但是这不是<html>乐于助人在没有喷砂的。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/55298fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 之前图案蚀刻，同样重要的是蚀刻整个样品的表面上。这是必要的，因为喷砂表面有许多镜面反射（ 见图6），其与斑点检测干扰。 <img alt="图6" src="/files/ftp_upload/55298/55298fig6.jpg" /> 图6:喷砂表面没有腐蚀。虽然不是作为原料的金属表面作为突出，相对较小的镜面反射（其中一些被突出显示黄色箭头）仍足以防止随后的斑点检测，所以没有校准可以与此目标进行。ARGET ="_空白"&gt;点击此处查看该图的放大版本。 以不同的速度施加激光给出不同的背景色。背景颜色起着圆圈和背景之间的对比度的显著作用。因此，至关重要的是要确定最佳的背景颜色。为此，为圆形的板衬托下的一组不同背景的创建（参见图7）。背景用的OpenCV 23，其被OpenCV的摄像机标定模块17中使用的特征检测模块进行测试。在这项工作中，目标被由不锈钢制成，因为它是在诊所的医疗器械中使用的最常见和最可靠的材料。这种材料是免费提供的，不贵，功能强大，且易于消毒。其它材料可能被用于校准的目标，如铝或碘化金属，但这是SCOP今后的工作电子邮件。 <img alt="图7" src="/files/ftp_upload/55298/55298fig7.jpg" /> 图7:不锈钢板具有与激光蚀刻不同的背景颜色的调色板。实际实验结合OpenCV的特征检测模块，以确定哪些背景色给出的斑点-背景对比度23方面的最佳结果进行的。左侧的内窥镜视图显示板。适中的背景颜色（即那些其他的暗和最亮的）在这个调色板中产生更好的斑点检测。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/55298fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 其中之一这项工作的优势在于执行使用编造目标校准需要2-3分钟。大部分努力去ES手动稳定内窥镜获得校准图案体面的看法。用一个定制的内窥镜保持器可以消除手动稳定化，这反过来又可以显著缩短校准时间的必要性。 <img alt="视频1" src="/files/ftp_upload/55298/55298video1.jpg" /> 视频1:视频显示失真如何光校准可以一起使用开发校准目标与endocal软件来执行。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/fetoscope-calibration-480.mov" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a> 相比格特等人的工作我们的工作的优点。 图15是OpenCV的照相机校正模块17可被用作为校准，而无需任何修改或定制parameterization。因为OpenCV的是一种行之有效的，并维护良好的软件包，并在计算机视觉界非常流行，用它消除了编写和维护定制软件的需求。为读者的方便，紧凑的GUI应用程序设置16，其读者可以容易地安装和使用，以测试新的校准目标。我们的方法的一个缺点相比格特等人的 图15是他们的方法是更健壮的图案的遮挡，因为它不要求所有斑点的检测。 最初，在棋盘图案的校准目标被制作为这个工作。然而，这种类型的校准目标的被证明是不合适的实验由于检测棋盘正方形的角上的困难。角检测依赖于基于直方图的图像二值化（见OpenCV的源代码24）。这小鬼在于为暗和亮方块，这可能不符合我们的棋盘图案被保证的，部分由于镜面反射，如在图6中所示的那些之间有明显的颜色对比的需要。这样的镜面反射，即使是背景后蚀刻存在;然而，圆的检测似乎是这个缺点不太敏感。 在当前的设置，只有校准目标的垂直的视图允许成功的斑点检测。这是由于从靶表面以倾斜角度妨碍斑点检测的镜面反射。我们正在进一步提高目标，以便允许在更宽的角度范围内，这可以潜在地改善进行校准20的质量获得的意见。 在实时胎盘拼先前提出的11管道，反式的计算形成映射图像对依赖于成功检测和功能分组。光学畸变，另一方面，使一组具有刚性几何特征出现跨越图像不同。因此，这种差异会导致在所计算的转换，这导致在所得到的图像马赛克漂移误差。由于最突出的光学畸变存在对边缘，内窥镜图像正在裁剪到心底深处的地区。一个良好的校正光学畸变会潜在地允许每个图像的较大部分到拼接过程中掺入。这种方法的优点是双重的。第一，这将增加的检测的特征的数目在每个图像中，潜在地提高了图像变换的计算。第二，这将允许在整个目标解剖表面在更短的时间内重建。

<html>摄像机标定是在已深入研究历年1，2，3计算机视觉领域的一个众所周知的问题。的摄像机标定程序的一个关键步骤是估计失真模型的参数，以及固有照相机参数，通过与来自照相机图像具有子像素精度的已知几何形状提取点的网格。与棋盘图案具有黑色和白色方块校准目标通常用于此目的。圆形斑点提供另一种图案4，5，6。 在最近几年，出现了在手术导航技术的发展为胎儿手术程序，诸如双胎输血综合征（TTTS）的治疗上胎儿越来越大的兴趣&gt; 7，8，9，10。作为视图的胎儿镜的场（ 即，在胎外科手术中使用的内窥镜）是非常有限的，用于映射的胎盘脉管系统，而无需使用外部跟踪的方法被提出，以帮助TTTS手术11，12，13。胎儿镜图像内的光学畸变对依赖于视觉信息提取11这些计算方法拼接不利影响。因此，存在对用于围可操作校准fetoscopes使得光学失真补偿可以实时干预期间进行成本有效和快速的工具未满足的需要。 由于这样的事实，该胎儿镜在介入期间浸没在羊水，一个之间的折射率差IR和羊水经典呈现在空中摄像机标定方法不适合胎儿手术程序。从在空气摄像机参数推定流体浸入相机参数是一个困难的任务，需要在流体浸入校准目标14中的至少一个图像。此外，围手术期，流体浸渍胎儿镜摄像机标定是由于灭菌要求和限制允许在手术室的材料目前是不切实际的。由于这些原因，校准内窥镜光学畸变通常不是目前临床工作流的一部分。在这个手稿的工作是试图通过设计和生产消毒和实用的光学畸变校正目标具有非对称的圆形图案，关闭这台相机标定的差距。先前，格特等。制作自定义校准装置具有氧化铝板作为校准目标。他们的甲基OD，然而，仅在与他们开发15定制校准算法相结合。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.2mm Metal sheet 316 Grade, 40 mm by 40 mm</td>
		</tr>
		<tr>
			<td>Water container at least 50mm by 50mm by 30mm</td>
		</tr>
		<tr>
			<td>A sterilisation package</td>
		</tr>
		<tr>
			<td>Seline water</td>
		</tr>
		<tr>
			<td>Manual metal cutter</td>
		</tr>
		<tr>
			<td>A file to round up the corners</td>
		</tr>
		<tr>
			<td>A wooden or metal block 50 mm by 50 mm at least 10 mm thick</td>
		</tr>
		<tr>
			<td>A vise (desirable but not required)</td>
		</tr>
		<tr>
			<td>Sand Blasting machine</td>
		</tr>
		<tr>
			<td>GUI application to create .dxf file with the pattern (https://github.com/gift-surg/endocal)</td>
		</tr>
		<tr>
			<td>PC</td>
		</tr>
		<tr>
			<td>Laser Cutter</td>
		</tr>
		<tr>
			<td>Autoclave</td>
		</tr>
		<tr>
			<td>An endoscope calibration software from GitHub (http://docs.opencv.org/2.4/doc/tutorials/calib3d/camera_calibration/camera_calibration.html)</td>
		</tr>
		<tr>
			<td>Endoscope</td>
		</tr>
		<tr>
			<td>OpenCV camera calibration module (https://github.com/opencv/opencv)</td>
		</tr>
	</tbody>
</table>

medical-grade sterilizable target for fluid-immersed fetoscope optical distortion calibration

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal Therapy and Surgery) project. One of the aims of this project is to engineer novel, real-time image processing methods for intra-operative use in the treatment of congenital birth defects, such as spina bifida and the twin-to-twin transfusion syndrome. The developed target allows for the sterility-preserving optical distortion calibration of endoscopes within a few minutes. Good optical distortion calibration and compensation are important for mitigating undesirable effects like radial distortions, which not only hamper accurate imaging using existing endoscopic technology during fetal surgery, but also make acquired images less suitable for potentially very useful image computing applications, like real-time mosaicing. In this paper proposes a novel fabrication method to create an affordable, sterilizable calibration target suitable for use in a clinical setup. This method involves etching a calibration pattern by laser cutting a sandblasted stainless steel sheet. This target was validated using the camera calibration module provided by OpenCV, a state-of-the-art software library popular in the computer vision community.

我们通过在喷砂不锈钢金属片，其设计如图1中所示的蚀刻不对称圆的图案创建的灭菌校准目标。显示了与胎儿镜一起行动这一校准目标的典范设置如图2。为了养活这个设计到激光蚀刻的软件，自定义应用程序在Python编程语言16开始实施。创建设计图案包括迭代地蚀刻在金属片平行线。用于图案具有在端一致的颜色，这些线之间的距离应该小于所述激光束的宽度（参见图1的插图） -这值是45微米的VIOLINO（LASERVALL）激光切割机。 <img alt="图1" src="/files/ftp_upload/55298/55298fig1.jpg" /> <st<html>荣&gt;图1:刻有花纹不对称的特色界的3×11格的设计。插图:放大视图不对称界的网格。线之间的距离为45微米（等于激光束的宽度），并且每个圆的直径为1毫米。其它尺寸可以用于网格为好，但这个被认为是最佳的相对于视胎儿镜场。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/55298fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55298/55298fig2.jpg" /> 图2:的Exemplar设置与使用的校准目标。水浸渍胎儿镜的尖端在右侧的校准目标被引导。在左边是英国一分钱规模提供信息。"目标="_空白"&gt;点击此处查看该图的放大版本。 所制造的校准目标允许检测用的OpenCV 17，其位置，然后分类到预定义的非对称的圆形网格的内窥镜视频流中的圆形图案（参见图3）。使用与已知的网格几何结合该信息，内部相机参数可被估计。这些包括相机矩阵和失真系数。相机基质由焦距和沿着二维图像平面的x轴和y轴的光学中心。失真系数是基于欠压康拉模式3。请注意，对于这项工作，只有径向畸变参数估计。对于理论的一个简短的讨论，与实际的例子，看到了OpenCV的摄像机标定模块的网页&lt;SUP类="外部参照"&gt; 17和MATLAB摄像机标定工具箱22。关于相机校准过程的更多细节都在张的工作提供20。该endocal软件库功能的编造校准目标16 10内窥镜意见的样本数据集。使用这种数据集，0.28个像素的平均重投影误差校准（分:0.16，最大值为0.45）获得。这相当于由格特等报道的0.25个像素。用他们的定制校准算法15。相同的研究组，但是，在15使用该方法用于校准用于胎盘拼接18的内窥镜照相机时报告在一个更近的纸张的0.6像素的重新投影误差。 <img alt="图3" src="/files/ftp_upload/55298/55298fig3.jpg" /> <st荣&gt;图3:校准模式的实时检测。从校准应用16的屏幕截图为特色的检测校准模式使用虚拟现实可视化从OpenCV的17叠加到实况视频流。请注意，校准图案的每一个检测的柱是由一个不同的颜色强调。所检测到的圆，与已知的几何形状相结合，被用于计算照相机参数。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/55298fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 估计的相机参数用于光学失真校正。 图4示出的矩形的棋盘图案，如使用胎儿镜，其中，光学畸变使行显示的曲线观看。注意，该线路中的distorti显示正常上校正后的图像。 <img alt="图4" src="/files/ftp_upload/55298/55298fig4.jpg" /> 图4:光学失真校正。从校准应用16具有从棋盘图案（左）与上述失真校正图象（右）一个胎儿镜记录实况视频图像的屏幕截图。三个示例性线绘制在两个图像中，每个从一个角到另一个，其中，该轨迹是线性的。由于光学畸变，这些行显示在原来的胎儿镜影像曲线。 <a href="http://ecsource.jove.com/files/ftp_upload/55298/55298fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration at JoVE.com

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

对于流体沉浸胎儿镜的光学畸变校正医用灭菌目标

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal ...

medical-grade-sterilizable-target-for-fluid-immersed-fetoscope

Research

JoVE Journal

Bioengineering

7.6K 次观看.  University College London. This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

视频: Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距"和"分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

生物医学植入物的石墨涂料

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

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 ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...