作者感谢琳达·特纳与微生物制剂的援助。 RZ和LGW是由罗兰大学，哈佛大学和CGB资金资助的短斗篷​​基金会的学者，科学无国界计划，巴西（进程＃7340-11-7）

1。设置和数据采集<ol><li>从冻结的股票增长22 链球菌菌株V4051-197（平滑游泳）细胞在KTY介质的文化。孵育在旋转摇床上过夜，在35℃和150rpm下，以饱和。 </li><li>接种10ml新鲜KTY培养基用500μl的饱和培养物。孵育另外的3.5小时，在35℃和150rpm下，直到细胞在λ= 600纳米（约5×10 8个细胞/ ml），达到约1.0的光密度。 </li><li>稀释1:400在新鲜培养基中，得到游动细胞的最终浓度。 </li><li>在显微镜载玻片上，使润滑脂的环（ 例如，从填充有凡士林注射器）约1毫米的高度，然后将一滴在中心的样品溶液。放置在顶部，轻压玻璃盖的边缘密封，确保液与两个滑动盖和玻璃接触。由此产生的样品室应该是阿柔ND 100-200微米的深度。 </li><li>将样品室中的盖玻璃朝下显微镜，并小心，以防止气泡的形成在油玻璃和透镜之间。 </li><li>重点放在样品室的底部表面的显微镜，并带来冷凝器进行对焦。 </li><li>关掉标准照明体和放置在LED头显微镜的聚光器孔径的后面。设置LED电源的最大输出。 </li><li>关闭冷凝器光圈到最大程度，并且如果需要，轻推在其安装的LED，直到照明的中心在上述物镜的数值孔径。 </li><li>切换电脑和相机，以及所有的光重定向到显微镜的相机端口。调整帧速率和图像尺寸的图像采集软件。 </li><li>使画面曝光时间越短越好，同时仍保持良好的对比度。检查图像亮度直方图，以保证图像不饱和特德或曝光不足。 </li><li>如果有必要，重新聚焦显微镜，让感兴趣的对象稍微散焦（通常为10-30微米）。在物体与焦平面应该在相同的培养基中（ 即焦平面应位于样品室室内）。 </li></ol> 2。重建处理数据的第一步骤是将数值重新调整视频帧在一系列不同的深度，产生一个堆栈的图像。用户友好的软件，这样做可以在这里找到: <a href="http://www.rowland.harvard.edu/rjf/wilson/Downloads.html">http://www.rowland.harvard.edu/rjf/wilson/Downloads.html</a>与示例图像（使用倒置显微镜获得的，和60X油浸物镜）和光线场景文件追踪渲染。 <ol><li>利益输入和帧其各自的背景为单独的图像文件，在该接口上的有关空格。背景图像是一帧使得fairly代表视频的背景下，在没有全息图，并且将用于抑制任何固定图案噪声可能干扰全息图的定位和分析。 </li><li>在运行程序之前将值输入到屏幕左侧的全局设置框。前三个是输出栈参数:第一帧的轴向位置（&#39;开始注重&#39;）;在重建切片堆叠数（&#39;步数&#39;）;各片之间在堆栈中的轴向距离（&#39;步长&#39;） 。 </li><li>为了重建与正确的比例的目的，步长应为相同的尺寸的横向的像素间距。在"像素/微米"框，光照波长和介质的折射率在过去两年框中输入摄像机的横向取样频率（1/pixel间距）。该程序的默认值适合重建的示例数据。 </li><li>按"翻转的Z-梯度"按钮，如果OBJE中心克拉是黑暗的全息图（见示例帧2005和讨论更多的细节部分）。 </li><li>检查带通滤波器开/关状态（默认为开启）。这个可选的带通滤波器，位于下方的渐变，翻转开关盒，负责抑制由噪声点的贡献。带通滤波器生成后立即应用到每一个图像切片。 </li><li>检查开/关状态（默认为上）中间输出开关。中间分析步骤都写在两个输出视频，未压缩的avi文件:第一是重新调整堆栈（文件名&#39;_stack.avi&#39;结尾），第二个是堆栈中的轴向梯度操作后（文件名以&#39; _gradient.avi&#39;）。理想情况下，这第二个堆栈将包含感兴趣的对象突出显示为对一个黑暗的背景明亮的物体。 </li><li>设置完成后在主窗口中的所有参数，按"运行"。选定的帧将出现在软件的主箱。 </li><li>使用工具栏将图像放大（左键）和缩小（按shift +左键）的左侧找到放大镜工具。使用工具栏上的矩形工具，选择感兴趣区域（ROI）的区域。捕捉尽可能多的全息图的边缘尽可能的矩形内，因为这将优化改造。 </li><li>按"进程"按钮，生成两个图像堆栈。检查使用ImageJ（可免费在获得由此产生的堆栈<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> ）。如果感兴趣的对象可以清楚地看出，在梯度图像的堆栈，请继续下一步要提取的对象的坐标。 </li></ol> 3。翻译<ol><li>除了框架重新调整，这个程序可以定位在X，Y，Z中感兴趣的对象的坐标为每个体素。按"特征提取"按钮来启用该功能。 </li><li>输入一个路径，包括扩展名（例如:c:家 output.inc），为输出坐标文件。在"输出坐标式"对话框中选择"POV-Ray的风格"。这将导致程序编写可以使用免费的POV-Ray的光线跟踪软件包（可从以下地址获得可视化的对象文件<a href="http://www.povray.org/">http://www.povray.org/</a> ）。 </li><li>重新处理图像作为上文第2节。该方案将提供一系列的（X，Y，Z）在选定的ROI坐标，在"输出坐标"框写入的文件名。物体坐标的提取需要显著长于堆栈生成。 </li><li>确保样品POV-Ray的文件（与重构代码）是在同一文件夹中刚刚生成的坐标文件。编辑范例。POV文件，并在引号替换文件名就行了 ＃包括"MYFILE.inc" 与由重建代码所产生的数据文件的名称。 </li><li>点击POV-Ray的的"运行"按钮，呈现一个位图图像。 Ŧ他的相机位置，灯光和纹理选项只是一些在POV-Ray的可自定义的，详情参见联机文档。 </li></ol>

<ol>
	<li>Xu, W., Jericho, M. H., Meinertzhagen, I. A., Kreuzer, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+in-line+holography+for+biological+applications.">Digital in-line holography for biological applications.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (20), 11301-11305 (2001).</li><li>Sheng, J., Malkiel, E., Katz, J., Adolf, J., Belas, R., Place, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+holographic+microscopy+reveals+prey-induced+changes+in+swimming+behavior+of+predatory+dinoflagellates.">Digital holographic microscopy reveals prey-induced changes in swimming behavior of predatory dinoflagellates.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 17512-17517 (2007).</li><li>Lee, S. H., Roichman, Y., 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=Characterizing+and+tracking+single+colloidal+particles+with+video+holographic+microscopy.">Characterizing and tracking single colloidal particles with video holographic microscopy.</a> Opt. Express. 15 (26), 18275-18282 (2007).</li><li>Fung, J., Martin, K. E., Perry, R. W., Katz, D. M., McGorty, R., Manoharan, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+translational,+rotational,+and+vibrational+dynamics+in+colloids+with+digital+holographic+microscopy.">Measuring translational, rotational, and vibrational dynamics in colloids with digital holographic microscopy.</a> Opt. Express. 19 (9), 8051-8065 (2011).</li><li>Lee, S. H., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+microscopy+of+holographically+trapped+three-dimensional+structures.">Holographic microscopy of holographically trapped three-dimensional structures.</a> Opt. Express. 15 (4), 1505-1512 (2007).</li><li>Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+digital+holographic+microscopy.">Principles and techniques of digital holographic microscopy.</a> SPIE Rev. 1, 018005 (2010).</li><li>Corkidi, G., Taboada, B., Wood, C. D., Guerrero, A., Darszon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+sperm+in+three-dimensions.">Tracking sperm in three-dimensions.</a> Biochem. Biophys. Res. Comm. 373, 125-129 (2008).</li><li>Santi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Sheet+Fluorescence+Microscopy+:+A+Review.">Light Sheet Fluorescence Microscopy : A Review.</a> J. Histochem. Cytochem. 59, 129-138 (2011).</li><li>van Blaaderen, A., Wiltzius, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Real-Space Structure of Colloidal Hard-Sphere Glasses. Science. 270, 1177-1179 (1990).</li><li>Besseling, R., Weeks, E. R., Schofield, A. B., Poon, W. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+Colloidal+Glasses+under+Steady+Shear.">Three-Dimensional Imaging of Colloidal Glasses under Steady Shear.</a> Phys. Rev. Lett. 99, 028301 (2007).</li><li>Schall, P., Weitz, D., Spaepen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Rearrangements+That+Govern+Flow+in+Colloidal+Glasses.">Structural Rearrangements That Govern Flow in Colloidal Glasses.</a> Science. 318, 1895-1899 (2007).</li><li>Rosen, J., Brooker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+incoherent+color+holography.">Fluorescence incoherent color holography.</a> Opt. Exp. 15, 2244-2250 (2007).</li><li>Jericho, M. H., Kreuzer, H. J., Kanka, M., Riesenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phase+and+refractive+index+measurements+with+point-source+digital+in-line+holographic+microscopy.">Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy.</a> Appl. Opt. 51 (10), 1503-1515 (2012).</li><li>Mudanyali, O., Erlinger, A., Seo, S., Su, T., Ozcan, D. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensless+On-chip+Imaging+of+Cells+Provides+a+New+Tool+for+High-throughput+Cell-Biology+and+Medical.">Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical.</a> J. Vis. Exp. 34, (2009).</li><li>Langehanenberg, P., Kemper, B., Dirksen, D., von Bally, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofocusing+in+digital+holographic+phase+contrast+microscopy+on+pure+phase+objects+for+live+cell+imaging.">Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging.</a> Appl. Opt. 47 (19), (2008).</li><li>Elliot, M. S., Poon, W. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+optical+microscopy+of+colloidal+suspensions.">Conventional optical microscopy of colloidal suspensions.</a> Adv. Coll. Interf. Sci. 92, 133-194 (2001).</li><li>Agero, U., Monken, C. H., Ropert, C., Gazzinelli, R. T., Mesquita, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+fluctuations+studied+with+defocusing+microscopy.">Cell surface fluctuations studied with defocusing microscopy.</a> Phys. Rev. E. 67, 051904 (2003).</li><li>Born, M., Wolf, 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> Principles of Optics, 6th Ed. , (1998).</li><li>Wilson, L., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Localization+of+weak+scatterers+in+digital+holographic+microscopy+using+Rayleigh-Sommerfeld+back-propagation.">3D Localization of weak scatterers in digital holographic microscopy using Rayleigh-Sommerfeld back-propagation.</a> Opt. Express. 20, 16735-16744 (2012).</li><li>Bohren, C., Huffman, 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> Absorption and Scattering of Light by Small Particles. , (1983).</li><li>Balaev, A. E., Dvoretski, K. N., Doubrovski, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractive+index+of+escherichia+coli+cells.">Refractive index of escherichia coli cells.</a> Saratov Fall Meeting 2001: Optical Technologies in Biophysics and Medicine III. 4707 (1), 253-260 (2002).</li><li>Berg, H. C., Manson, M. D., Conley, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+Energetics+of+Flagellar+Rotation+in+Bacteria.">Dynamics and Energetics of Flagellar Rotation in Bacteria.</a> Symp. Soc. Exp. Biol. 35, 1-31 (1982).</li><li>Garcia-Sucerquia, J., Xu, W., Jericho, S. K., Klages, P., Jericho, M. H., Kreuzer, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+in-line+holographic+microscopy.">Digital in-line holographic microscopy.</a> Appl. Optics. 45 (5), 836-850 (2006).</li><li>Restrepo, J. F., Garcia-Sucerquia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnified+reconstruction+of+digitally+recorded+holograms+by+Fresnel-Bluestein+transform.">Magnified reconstruction of digitally recorded holograms by Fresnel-Bluestein transform.</a> Appl. Optics. 49 (33), 6430-6435 (2010).</li><li>Dubois, F., Joannes, L., Legros, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+three-dimensional+imaging+with+a+digital+holography+microscope+with+a+source+of+partial+spatial+coherence.">Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence.</a> Appl. Optics. 38 (34), 7085-7094 (1999).</li><li>Kanka, M., Riesenberg, R., Petruck, P., Graulig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+(NA=0.8)+in+lensless+in-line+holographic+microscopy+with+glass+sample+carriers.">High resolution (NA=0.8) in lensless in-line holographic microscopy with glass sample carriers.</a> Opt. Lett. 36 (18), 3651-3653 (2011).</li><li>Dubois, F., Requena, M. L., Minetti, C., Monnom, O., Istasse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+spatial+coherence+effects+in+digital+holographic+microscopy+with+a+laser+source.">Partial spatial coherence effects in digital holographic microscopy with a laser source.</a> Appl. Optics. 43 (5), 1131-1139 (2004).</li><li>Magariyama, Y., Sugiyama, S., Muramoto, K., Kawagishi, I., Imae, Y., Kudo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+bacterial+flagellar+rotation+rate+and+swimming+speed.">Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.</a> Biophysical Journal. 69 (5), 2154-2162 (1995).</li><li>Berg, H. C., Brown, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+Escherichia+coli+analysed+by+three-dimensional+tracking.">Chemotaxis in Escherichia coli analysed by three-dimensional tracking.</a> Nature. 239 (5374), 500-504 (1972).</li><li>Brooker, G., Siegel, N., Wang, V., Rosen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+resolution+in+Fresnel+incoherent+correlation+holographic+fluorescence+microscopy.">Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy.</a> Opt. Express. 19 (6), 5047-5062 (2011).</li></ol>

作者宣称，他们有没有竞争的财务权益。

在这个实验方案中最重要的步骤是从一个稳定的实验装置的图像的精确捕捉。差的背景资料，高保真重建几乎是不可能的。同样重要的是，避免物镜与内部相位对比元件（如通过物镜的背面看时暗环形可见的），因为这样可以降低重建图像。感兴趣的对象应该是远远不够的焦平面那几双衍射条纹的可见其形象（一个合适的启发是，散焦图案应具有焦点的对象的10倍线性尺寸 - 见示例数据）。物体?...

内嵌数字全息显微镜（DIHM）允许显微镜的样品，如游泳微生物1,2和软物质系统3,4，以最小的修改一个标准的显微镜装置的快速三维成像。本文DIHM的教学演示提供，围绕在我们的实验室开发的软件中心。本文包括如何设置显微镜，优化数据采集，处理和记录的图像重建三维数据的描述。该软件（部分基于软件由DG格里尔和其他5开发）例如图像，且可自由查看我们的网站上。下面说明提供的配置显微镜，从全息图重建三维体积和使用一个免费射线跟踪软件包呈现所得的感兴趣体积所必需的步骤。本文总结了影响重建质量因素的讨论和DIHM与竞争方法进行了比较。 虽然DIHM被描述前一段时间（对于其原则和发展进行全面检讨，见金6），所需要的计算能力和图像处理专业技术迄今在很大程度上限制了它的使用，以专门研究小组，重点放在仪器的发展。这种局面正在改变在计算机和摄像头技术的最新进展的光。现代桌面电脑可以轻松应对处理和数据存储需求; CCD或CMOS摄像头存在于大多数显微镜实验室，以及必要的软件被免费提供在互联网上通过谁在开发该技术投入时间组。 各种方案已经被提出用于成像微观对象的配置中的三维样品体积。许多这些扫描技术7,8，其中一个堆栈的图像的记录由米通过样品echanically平移所述图像平面。激光扫描共聚焦荧光显微镜也许是最熟悉的例子。通常，荧光染料被添加到相位物体，以便实现样品的对比可以接受的水平，并在共聚焦装置用于空间定位的荧光发射。这种方法导致了显著的进步，例如在胶体科学它允许访问拥挤的系统9-11的三维动态。使用标签是荧光共聚焦显微镜和DIHM之间的一个重要差别，但两种技术的其他功能都值得比较。 DIHM具有显著速度上的优势在于，该装置没有移动部件。在共聚焦系统的机械扫描镜放置一个上限的数据采集率 - 通常大约30帧/秒的512×512像素的图像。可以通过物理译获得一摞这样的图像从不同的焦平面阿婷帧之间的样品台或物镜，导致每秒大约一体积为30帧堆栈的最终捕获率。在比较的基础上，现代CMOS相机的全息系统可以捕获2000帧/秒在相同的图像尺寸和分辨率，每个帧被`离线&#39;处理，得到样品体积的独立快照。重申一下:不需要荧光样品DIHM，虽然系统已经开发出来，进行荧光主体12的全息重建。以及三维体积信息，DIHM也可以被用来提供定量相衬图像13，但是这超出了本文的讨论范围之内。 原始DIHM数据图像是二维的，并且在某些方面看起来像标准的显微镜图像，虽然失焦。 DIHM和标准亮视野显微镜之间的主要区别在于，围绕物体的衍射环我n个视场，这些都是因照明的性质。 DIHM需要比亮场更连贯源 - 通常是LED或激光。在全息图上的衍射环含有重构的三维图像所需的信息。有两种主要的方法来解释DIHM数据，直接拟合和数值重新调整。第一种办法是适用的地方衍射花纹的数学形式事先已知3,4案件;这种情况被通过极少数样球体，缸，一半平面障碍物的简单对象遇见。直接拟合也可以适用于该对象的轴向位置是已知的情况下，图像可以使用的图像模板14中的查找表被安装。 第二种方法（数字重聚焦）是较为普遍，并依赖于使用的衍射环在二维全息图图像进行数值重构光场在一些（任意间隔）焦平面整个样品体积。这样做的6几个相关的方法存在，这项工作采用瑞利-索末菲反向传播技术所描述的李和格里尔5。这个程序的结果是一个栈，复制手动更改显微镜焦平面（因此得名&#39;数值重新调整&#39;）的效果的图像。一旦已经生成了一个堆栈的图像，必须获得在焦量被摄物体的位置。一个数字图像分析的启发式，如局部强度方差或空间频率内容的，已经呈现给量化聚焦的锐度在不同点的样品15中。在每一种情况下，当一个特定的图象量度被最大化（或最小化），该对象被认为是在焦点上。 不像其他的计划，寻求以识别特定的焦平面其中一个对象是"焦点"，在这项工作中的方法挑选输出Points的谎言感兴趣的对象内部，从而在广泛焦平面延伸。这种方法可以适用于广泛的主题，并且特别适合于延伸，弱散射样品（相位物体），如杆状胶体，细菌的链条或真核生物的鞭毛。在这样的样品，当对象穿过焦平面的图像的对比度改变;散焦图像具有光中心，如果该对象是在焦平面与暗中心的一侧上，如果它是在另一。纯相的对象几乎没有任何相反，当他们躺在正是在焦平面。这种现象对比反转的已经由其他作者16,17讨论，并最终由于的Gouy相位异常18。这已被放置在全息的上下文中更严格的立足点别处，其中所述技术的限制进行评估;在位置的典型的不确定性是150纳米（约一个像素）在EAC的顺序H方向19。古埃相位异常的方法是用于确定在三维空间中扩展的对象的结构的几个定义良好DIHM方案之一，但尽管如此某些对象是有问题的重构。直接位于沿着光轴（指着相机）的对象是难以精确地重建，在该对象的长度和位置误差变大。这种限制是部分由于受限制的位深度的记录全息图（不同的灰度级，该相机可以记录的数量）的像素。当感兴趣的对象是非常接近焦平面另一个有问题的配置发生。在这种情况下，对象的实际和虚拟图像重建接近，到复杂的光学领域都是难以解释而产生。第二，稍不那么重要这里关注的是，所得的衍射条纹占用更少的图像传感器，而这种粗粒INFormation导致较差质量的重建。 在实践中，一个简单的梯度滤波器被应用到三维重构的体积来检测强度强反转沿照射方向。地区，强度变化很快从亮到暗，或反之亦然，然后与散射区域相关联。弱散射物体有很好的描述为这样的元素20的非相互作用集合，这些个人缴费之和为总的散射场是利用瑞利-索末菲反向传播方法容易倒。在本文中，轴向强度梯度技术被应用到一个链链球菌细胞。细胞体是相位对象（物种大肠杆菌具有测量21为1.384在波长λ= 589nm的折射率;的链球菌菌株很可能是类似的），并在显示为连接小圆块的高强度链日渐变滤镜后É样品体积得到了应用。适用于该过滤体积标准阈值和特征提取方法允许容积像素（体素）相对应的区域内的细胞内的提取。这种方法的一个特别的优点是它允许在轴向方向上的物体的位置的明确的重建。类似的方法（至少，那些记录的全息图邻近的对象，通过显微镜的物镜成像）患有无法确定此位移的符号 。虽然瑞利 - 索末菲重建方法还签署无关在此意义上，梯度操作允许我们上面和下面的焦平面弱相位物体之间的区分。

<table><tbody><tr><td>Nikon Eclipse Ti-E inverted microscope</td><td>Nikon Corp.</td><td></td><td>&amp;nbsp;</td></tr><tr><td>LED</td><td>Thorlabs</td><td>M660L3</td><td>emission wavelength &amp;lambda;=660 nm, linewidth approximately 20 nm&amp;nbsp;</td></tr><tr><td>LED power supply</td><td>Thorlabs</td><td>LEDD1B</td><td>&amp;nbsp;</td></tr><tr><td>Thread Adapter</td><td>Thorlabs</td><td>SM2T2</td><td>&amp;nbsp;</td></tr><tr><td>Thread Adapter</td><td>Thorlabs</td><td>SM1A2</td><td>&amp;nbsp;</td></tr><tr><td>Frame Grabber board</td><td>EPIX</td><td>PIXCI E4</td><td>&amp;nbsp;</td></tr><tr><td>High-Speed CMOS camera</td><td>Mikrotron</td><td>MC-1362</td><td>&amp;nbsp;</td></tr></tbody></table>

digital inline holographic microscopy (dihm) of weakly-scattering subjects

弱散射的物体，诸如小的胶体粒子和大多数生物细胞中，经常遇到的显微镜。的确，已经开发了一系列的技术，以便更好地可视化这些相位物体;相衬和DIC是最流行的方法用于增强对比度。但是，在超出成像平面的方向上的记录位置和形状仍然具有挑战性。本报告介绍了一种简单的实验方法精确地确定位置和在三维空间中的物体的几何形状，采用数字内嵌全息显微镜（DIHM）。从广义上讲，可访问的样品体积是由相机传感器尺寸在横向方向上，并且在轴向方向上的照明相干性来定义。典型样品体积范围从200 微米 ×200 微米 ×200微米采用LED照明，以5毫米 ×5 毫米 ×5毫米或更大的用激光照射。这样的照明光被配置成使得平面波入射到样品上。在样品体积的物体然后散射光，这与未散射的光干涉而形成的干涉条纹垂直于所述照明方向。该图中（全息图）包含所需的三维重建的深度信息，并且可以在标准的成像设备，诸如CMOS或CCD照相机上被捕获。瑞利 - 索末菲反向传播方法被用来数值重新聚焦显微镜图像，并基于所述的Gouy相位的简单成像启发式异常是用来识别散射重构的体积内的对象。这个简单而可靠的方法导致对象的位置和形状的显微样品中的一个明确的，无模型测量。

为了演示DIHM的能力，实验是在一个链链球菌细菌进行。链本身测量10.5毫米长，是由6-7球柱面单元（2所述细胞在链轮的接近分割），直径范围为0.6-1微米。 图1a和1b示出了主接口重建和渲染软件。数字重聚焦过程的例子如图2所示，其中一个空间带通滤波器已被应用， 图3示出了梯度滤波器对图2中的图像的效果。用?...

Watch this Scientific Journal Video about Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects at JoVE.com

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

弱散射物体的三维位置可被唯一地识别利用数字内嵌全息显微镜（DIHM），它涉及到一个小的修改，以一个标准的显微镜。我们的软件使用加上瑞利 - 索末菲反向传播，得到三维位置和微观相对象的几何形状的简单的成像启发式。

的弱散射主题内嵌数字全息显微镜（DIHM）

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

digital-inline-holographic-microscopy-dihm-weakly-scattering

Research

JoVE Journal

Biology

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

12.2K 次观看.  Harvard University. 弱散射物体的三维位置可被唯一地识别利用数字内嵌全息显微镜（DIHM），它涉及到一个小的修改，以一个标准的显微镜。我们的软件使用加上瑞利 - 索末菲反向传播，得到三维位置和微观相对象的几何形状的简单的成像启发式。 

视频: Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

片上的无透镜荧光显微镜

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

科研

生物工程

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

紧凑的镜头少数字全息显微镜的MEMS检测与表征

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

工程学

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 ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

Hyperlens 综合显微镜和超分辨率成像的演示

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

免疫与感染

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

基于二维Gabor滤波器的光散射显微镜

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

生物学

Uncovering Hidden Dynamics of Natural Photonic Structures Using Holographic Imaging

In this method, the potential of optics and holography to uncover hidden details of a natural system's dynamical response at the nanoscale is exploited. In the first part, the optical and holographic studies of natural photonic structures are presented as well as conditions for the appearance of the photophoretic effect, namely, the displacement or deformation of a nanostructure due to a light-induced thermal gradient, at the nanoscale. This effect is revealed by real-time digital holographic interferometry monitoring the deformation of scales covering the wings of insects induced by temperature. The link between geometry and nanocorrugation that leads to the emergence of the photophoretic effect is experimentally demonstrated and confirmed. In the second part, it is shown how holography can be potentially used to uncover hidden details in the chemical system with nonlinear dynamics, such as the phase transition phenomenon that occurs in complex oscillatory Briggs-Rauscher (BR) reaction. The presented potential of holography at the nanoscale could open enormous possibilities for controlling and molding the photophoretic effect and pattern formation for various applications such as particle trapping and levitation, including the movement of unburnt hydrocarbons in the atmosphere and separation of different aerosols, decomposition of microplastics and fractionation of particles in general, and assessment of temperature and thermal conductivity of micron-size fuel particles.

The paper is primarily focused on the combined power of optical (linear and nonlinear) and holographic methods used to reveal phenomena at the nanoscale. The results obtained from the biophotonic and oscillatory chemical reactions' studies are given as representative examples, highlighting holography's ability to reveal dynamics at a nanoscale.

使用全息成像揭示自然光子结构的隐藏动力学

In this method, the potential of optics and holography to uncover hidden details of a natural system's dynamical response at the nanoscale is exploited. ...

化学

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

构建用于扩展视场单分子成像的高倾角扫描磁贴显微镜的指南

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.