在欧文和Cherna莫斯科维茨中心在魏茨曼科学研究所的纳米生物纳米成像进行了研究。 我们感谢她帮助设计和运行此协议Orna Yeger。

1。样品制备<ol><li>矿化组织提取组织进行审查后，可放置在仪器和影像。形象鼠标股骨应遵循以下步骤： <ol><li>从C57/Bl6胚胎18.5天postceutus（E18.5），取出后验尸腿。 </li><li>密封聚苯乙烯枪头采用环氧树脂或其他胶水（20-200μL）较窄的一端，并填写工作缓冲液（PBS或其它）的提示。 </li><li>适合紧到尖端的腿和密封封口膜表的另一端。 </li><li>枪头放入一个合适的人，并按照该协议第3章。对于可视化的鼠标腿胚胎股骨，该仪器是集40千伏和200μA。对于8μ分辨率1000 4倍的放大倍率的投影图像被收购。 </li></ol></li><li>非矿化组织已被初步固定，染色，以增加使用的许多8,9可用的协议之一的利益组织X射线衰减。对大鼠肺组织和类似的样品，准备协议是： <ol><li> Orthotopical植入裸大鼠肺非小细胞肺癌（NSCLC）NCI - H460 </li><li>肺癌结节开始被检出植入4周</li><li>牺牲老鼠，并立即用肝素生理盐水溶液混合，注入</li><li>注入左心室染色的支气管循环与稀释液，一个Microfil（Flowtech）（2毫升的复合溶液，3毫升稀释剂和固化剂0.3毫升） </li><li>提取物对大鼠的肺和心脏</li><li>装修成50毫升塑料试管，它紧紧地固定样本（见议定书第2章） </li><li>创建布乙醇阻尼管的底部放置一个饱和的乙醇气氛</li><li>胶水或螺丝管，在仪器的持有人</li><li>继续设置成像参数（第3章）。大鼠肺的完整影像源是设置在40KV和100μA。为了达到16μ的决议之一，已收购的0.5倍的放大倍率2500投影图像。 </li></ol></li></ol> 2。样品固定分辨率高，重要的是要避免在测量过程中的样品位置的任何改变。对于这一点，样品是紧紧固定到一个适合其规模的塑料收件人。聚苯乙烯枪头，塑料巴氏吸管或特制的塑料持有人在这方面使用。根据实验要求，可检查样品在空气中，或沉浸在乙醇或缓冲溶液。典型的固定和最终定位仪器中的小鼠胚胎的腿是在图1所示。 <img src="/files/ftp_upload/2688/2688fig1.jpg" alt="图1"/> 图1。最后定位在小鼠胚胎显微CT仪器腿。 3。设置采集参数：X -射线电压和电流，CCD曝光时间<ol><li>样品放入持有人，把仪器的旋转舞台</li><li>第一个X -射线图像拍摄与电压和电流的任意设置。 </li><li>如果图像太暗，应先增加光子数，所以增加一点点的电流。如果这是不够的，应略有增加X射线光子的能量，即X射线管电压。 </li><li>如果图像太亮，应先降低电压，那么当前的。 </li><li>通过分级，可以增加图像的亮度。 1分级考虑到图像中的每个像素的强度，而2的分级需要每个2x2的像素矩阵的总和。图像将被约4倍，比在分级1的情况下更亮，但将有一半的决议。 </li><li>设置最佳的亮度后，一个优化相机妥协一侧和另一侧的实验的合理工期之间的最佳的对比度的曝光时间。 </li><li>的图像，尤其是低吸收样品的对比，可以提高通过使用过滤器，从而降低主要是低能量的光子，光子通量，。 </li></ol> 4。样品定位<ol><li>选择工作的放大倍率。可能的选择是0.5X，4X，10X，20X和40X。视野随着增加放大倍数。 </li><li>通过设置之间的X射线源和样品，样品和探测器之间的距离，获取最佳分辨率和实地查看。增加采样距离源头上减少视野，提高了分辨率。样品到探测器的距离，具有相反的效果。 </li></ol> 在3D观看整场应在目前的在各个角度的投影图像。应检查样品在不同的角度和旋转，使样品尽可能靠近旋转轴。为此，应该遵循以下步骤： <ol start="3"><li> 0度的图像，然后在-20度的旋转样品。如果想要的音量横向转移，一要正确重新定位旋转轴的位置。 </li><li>校正后，样本是在另一个角度的旋转和位置再次纠正，直到感兴趣的领域内的图像是从-90到90度的所有角度。 </li></ol> 5。高决议断层扫描<ol><li>在测量过程中，样本轮换一次一个小角度，在各个角度的投影图像是。图像的总数始终是一方所需的分辨率和时间的测量和另一侧的文件大小之间的妥协。如图2所示，每一个投影包括样品中的所有切片叠加在另一之一，因此不能透露样品的三维结构。 </li></ol><img src="/files/ftp_upload/2688/2688fig2.jpg" alt="图2"/> 图2。对大鼠肺（A）45 °（B）和90 °（C）旋转角度0 °的投影图像。 <ol start="2"><li>只有服用后至少介于-90和90度的投影图像，可以进行重建的样本量。重建需要10分钟和2小时之间，这取决于所使用的软件和的预测数。同样，最终的三维图像质量所需的分辨率和人愿意花时间和所产生的文件的大小之间的妥协。 </li></ol> 6。图像标度校准在重建图像的像素水平（值），形象独特。为了比较两个不同的图像，独特的强度等级要强加给每个图像。对于这个<ol><li>使用相同的实验条件为样本，运行与一个标准的幻象断层</li><li>重新校准使用的幻像中获得的值的样本图像。规模最常见的是豪森菲尔德（或CT）的规模。对于4倍的放大倍率15000背景值（水或PBS）被替换为0，最大值35000骨豪森菲尔德3000标准值取代。其他像素值从线性内插或外推基于这些限制。 </li></ol> 7。图像处理和分析在获得高清晰度的图像，提取相关信息，利用图像分析软件。要使用该软件包已被设计为工作具有非常大的文件（最高的20Gb）的。 8。代表性的成果一个从C57/Bl6鼠标代表了股骨在胚胎18.5天（E18.5） - 四天后开始矿化过程如图3所示。矿物层清晰可见（白色），而软组织在这个准备是不可见的。我们把1000的4倍线性放大投影图像。最后决议是8微米。一个体绘制图1所示的仔细的分析，表明，骨体积分数（矿化组织占领的骨体积分数）为0.18，和骨密度是723毫克/ 厘米 3 。这些价值观让我们比较有骨这种结构在其他的发展阶段。 <img src="/files/ftp_upload/2688/2688fig3.jpg" alt="图3"/> 图3。一个鼠标股骨胚胎的三维图像的不同表示。横向（横截面）（一），矢状（德尔梅迪奥横向）（b）条和快照卷的渲染（三）所示。 图4显示了一个女裸大鼠（RNU），12周龄原位植入与非小细胞肺癌（NSCLC）NCI - H460，肺的三维图像。 2500投影图像与线性放大了0.5倍，确保了16微米的最终解决。图像显示Microfil染色的血管（直径为20微米的），图像分析显示，植入4周后，多个癌结节形成。它们涵盖了相当一部分的肺容积（17％）。大多数肺染色发现在肿瘤的周边地区。值得注意的是，在图4b所示，几个血管目前还结节内，占地约3％的体积，据初步分析。 <img src="/files/ftp_upload/2688/2688fig4.jpg" alt="图4"/> 图4。癌结节的三维图像，在大鼠增长肺。容积再现（A）和卷（二）通过部分快照所示。癌结节用箭头标出。 电影1卷呈现在图1的小鼠股骨<a href="/files/ftp_upload/2688/2688movie1.mpg">。点击这里观看电影</a> 。 电影2卷图2中的大鼠肺的渲染<a href="/files/ftp_upload/2688/2688movie2.mpg">。点击这里观看电影</a> 。 电影3。通过肺部的串行部分。结节出现在大部分的片灰色地带<a href="/files/ftp_upload/2688/2688movie3.mpeg">。点击这里观看电影</a> 。

<ol>
	<li>Schambach, S. J., Bag, S., Schilling, L., Groden, C., Brockmann, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+micro-CT+in+small+animal+imaging.">Application of micro-CT in small animal imaging.</a> Methods. 50, 2-13 (2010).</li><li>Bauer, J. S., Link, T. M. <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+osteoporosis+imaging.">Advances in osteoporosis imaging.</a> Eur J Radiol. 71, 440-449 (2009).</li><li>Chappard, D., Retailleau-Gaborit, N., Legrand, E., Basle, M. F., Audran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+insight+bone+measurements+by+histomorphometry+and+microCT.">Comparison insight bone measurements by histomorphometry and microCT.</a> J Bone Miner Res. 20, 1177-1184 (2005).</li><li>Muller, R., Van Campenhout, H., Damme, B. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+human+bone+biopsies:+a+quantitative+structural+comparison+of+histological+sections+and+micro-computed+tomography.">Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography.</a> Bone. 23, 59-66 (1998).</li><li>Mueller, K., Yagel, R., Wheller, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Aliased+3D+Cone-Beam+Reconstruction+Of+Low-Contrast+Objects+With+Algebraic+Methods.">Anti-Aliased 3D Cone-Beam Reconstruction Of Low-Contrast Objects With Algebraic Methods.</a> IEEE Transactions on Medical Imaging. 18, 519-537 (1999).</li><li>Kachelriess, M., Schaller, S., Kalender, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+single-slice+rebinning+in+cone-beam+spiral+CT.">Advanced single-slice rebinning in cone-beam spiral CT.</a> Med Phys. 27, 754-772 (2000).</li><li>Endo, M., Komatsu, S., Kandatsu, S., Yashiro, T., Baba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination-weighted+Feldkamp-based+reconstruction+algorithm+for+cone-beam+CT.">A combination-weighted Feldkamp-based reconstruction algorithm for cone-beam CT.</a> Phys. Med. Biol. 51, 3953-3965 (2006).</li><li>Marxen, M., Thornton, M. M., Chiarot, C. B., Klement, G., Koprivnikar, J., Sled, J. G., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Med Phys. 31, 305-313 (2004).</li><li>Plourabou&#233;, F., Cloetens, P., Fonta, C., Steyer, A., Lauwers, F., Marc-Vergnes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+high-resolution+vascular+network+imaging.">X-ray high-resolution vascular network imaging.</a> J Microsc. 215, 139-148 (2004).</li></ol>

C57/Bl6小鼠在胚胎18.5天（E18.5）是四天后的矿化过程。在这一发展阶段，未来的骨是由许多层矿化osteoids，在图3清楚地看到。在这一点上，应该强调，矿化组织可以在较低的分辨率的可视化与不同的乐器，它需要较少的样品处理。本协议，除了提供更高的分辨率（微CT仪器在使用），提供最高的灵活性，为用户选择最佳的几何参数测量。 在图4的结果表明，在原位肺癌的动物模型，人类非小细胞肺癌可诱发血管和新生血管形成的招聘。我们认为，肺组织既不是感动，也没有在测量过程中改变其形状。用户应采取特别的预防措施，以避免这样的变化在一个断层。对于某些样品，特别是对软组织，建立特殊的设备保持完美的样品在测量过程中，固定。不幸的是，在肿瘤周围存在高的造影剂泄漏，防止周围血管可靠的量化。因此图像沾染一些染色剂，特别是在边缘，这显然是电影中的2和3。我们无法防止这种泄漏，但有用的信息，包括其大小，形状和存在内在的血管有关的癌结节并没有受到影响。我们可以清楚地得出结论，至少在这里进行了研究支气管流通，参与周围的血液供应肿瘤灌注，灌注一些肿瘤内也。

进行图像采集，我们已经使用了一个微型XCT - 400 microfocussed X射线层析成像系统，康科德，美国Xradia公司生产的。 图像处理和分析使用ImageJ（美国国立卫生研究院，美国），Avizo器（VSG，法国）和微视（通用电气公司，美国）软件包。任何可用的图像分析软件，可以用来代替

high resolution 3d imaging of ex-vivo biological samples by micro ct

非破坏性的量可视化可以实现，只有通过层析成像技术，其中最有效的是X射线微计算机断层扫描（μCT）。 高分辨率μCT是一个非常灵活而又准确的的3D考试前体内的生物样品 1，2（1-2微米分辨率）技术。相对于电子断层扫描，μCT允许4厘米厚的样品的检查。这种技术只需要测量的几个小时相比，在组织学周。此外，μCT不依赖于2D视学模型，从而可以补充，在某些情况下甚至可以取代组织学方法，3，4，这是既费时和破坏性。 μCT样品调节和定位非常简单，不需要高真空或极低的温度，这可能会产生不利影响的结构。样品定位和旋转180 °或360 °之间的一个microfocused的X射线源和探测器，其中包括一个闪烁和准确的CCD相机，对于每一个二维图像的角度，然后使用一个重建整个卷不同的可用算法5-7。 3D分辨率的增加与减少旋转步。本视频协议显示在高分辨率成像样品的准备，固定和定位的主要步骤。

Watch this Scientific Journal Video about High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT at JoVE.com

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

非破坏性的量可视化可以实现，只有通过层析成像技术，其中最有效的是X射线微计算机断层扫描（CT）。

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

high-resolution-3d-imaging-of-ex-vivo-biological-samples-by-micro-ct

Research

JoVE Journal

Bioengineering

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

18.6K 次观看.  Weizmann Institute of Science. 非破坏性的量可视化可以实现，只有通过层析成像技术，其中最有效的是X射线微计算机断层扫描（CT）。

视频: High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

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 ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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 ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...