部分支持这项工作是由格兰特没有。 213/09从以色列科学基金会，授予没有。 2005258从BSF的基础上，授予没有。 201665从欧洲研究委员会（ERC），由罗素Berrie在Technion工业的纳米技术研究所。我们承认Schulich商学院在Technion工业有关的供应和处理蓝藻的化学教授诺姆ADIR和法里斯萨拉梅的帮助。 Technion工业微纳米加工单位的帮助和支持斯韦特兰娜Yoffis是极大的赞赏。

1。 ESR的显微成像概述首先，我们提供一个简要的解释的ESR，血沉显微镜，和我们的系统的各个组成部分，然后我们将描述实际的成像实验。 电子自旋共振光谱技术中，在特定频率的电磁辐射吸收与未成对电子的自旋的分子，放在下一个外部的静磁场（图1）。 ESR是在广泛领域的科学，如化学，生物，物理，材料科学，聘用，自由基与顺磁中心的检测和鉴定。它是一种顺磁性分子在活体物种的研究环境的强大方法，并提供有关酸度（PH），粘度，氧和活性氧浓度 3的信息。 异构样品，ESR光谱信息，可以得到在空间分辨的方式（即通过获取图像），通过利用磁场梯度 4 。这是非常相似的核磁共振成像（MRI），更常用的方法主要是观察质子的自旋。截至目前为止，如ESR成像技术被应用于几厘米的规模比较大的，在毫米级分辨率的活标本。 （例如，参见图2，参考文献5。）ESR成像较近期的发展寻找小动物在毫米级分辨率毫米波和亚毫米大小的样品测量其功能扩展微米级的分辨率。此字段被称为ESR显微镜，今天可以提供三维ESR的图像接近1微米（参见图3中的有代表性的例子）的决议。 ESR的显微镜是一个传统的ESR光谱仪基本相似。它有一个产生静电场，一个微波系统的自旋激发和信号检测，拿着样品探头，和一个计算机化的控制台来控制采集过程和数据处理的磁铁。其他元件独特的ESR成像和现有的ESR microcopy也梯度磁场源，是电子系统的一部分，和梯度线圈，成像探头位于。我们的特定系统的显示更多的细节在协议中的电影和参考文献2中所述。 2。 ESR显微成像样品制备这一阶段描述的ESR显微成像实验样品制备方法。在这个阶段的细胞结束被放置在一个专门准备的玻璃ESR镜样品容器的底部，连同一个三苯甲基自由基6缓冲液。本协议描述了测量蓝藻细胞，因此，对于其他类型的细胞，适当调整可能需要在样品制备阶段。 <ol><li>首先，几个平方〜400 400微米大小的吸水纸，并插入到Eppendorf管中，随后充满蓝藻悬浮1.2毫升（浓度40毫克/毫升）。 </li><li>停牌是在6000转速在微量离心2分钟。 </li><li>在此之后，上清缓冲区完全删除除外〜50μL左避免蓝藻脱水。作为这一进程的结果，现在是饱和的吸水纸蓝藻细胞。 </li><li>用细镊子，少数纤维是从文件中提取并置于杯专门准备的玻璃样品架7底部。以下3 BG - 11解决方案8，9毫米的三苯（见图1）添加罚款注射器援助样品架。持有人，然后采用紫外光固化胶密封，留下一个小风口开放。 <table border="1"><tbody><tr><td> 库存4 </td><td> 股票3 </td><td> 股票2 </td><td> 库存1 </td></tr><tr><td> H 3 BO 3 2.86g/liter </td><td> K 2 HPO 4：3H 2 O 4.0g/liter </td><td> 硫酸镁 ： 7H 2 O 7.5g/liter </td><td>那2毫克 EDTA 0.1g/liter </td></tr><tr><td> MnCl 2：4H 2 O 1.81g/liter </td><td></td><td></td><td>铁铵柠檬酸0.6g/liter </td></tr><tr><td> 硫酸锌：7H 2 O 0.222g/liter </td><td></td><td></td><td>柠檬酸：1H2O 0.6g/liter </tD&gt; </tr><tr><td> 4：硫酸铜5H 2 O 0.079g/liter </td><td></td><td></td><td>氯化钙2 · 2H 2 O 3.6g/liter </td></tr><tr><td> COCl 2 ：· 6H 2 O 0.050g/liter </td><td></td><td></td><td></td></tr><tr><td> NaMoO 4 · 2H 2 O 0.391g/liter </td><td></td><td></td><td></td></tr></tbody></table> 计划1。BG - 11培养基的制备。 </li></ol> 3。 ESR显微成像实验<ol><li>要开始了成像实验，打开的ESR显微成像系统和谐振器内成像探头插入样品。 </li><li>现在，使用计算机控制软件，设置系统的“调教”模式，并找到共振微波频率的探头，将使用的ESR测量。 </li><li>以下的价值相匹配应用的微波频率，设置静态磁场，脉冲序列的时序参数和观察的ESR信号，以确保系统的功能和样品是精心准备。 </li><li>然后，将成像参数，如像素数，梯度的力量，和其所需的值的梯度脉冲的长度。 </li><li>以下设置，收集interpulse分离，价值观500，600，700 NS一个哈恩回波成像脉冲序列（图4）三个ESR的3D图像。 </li><li>样品投射灯是根据所需的实验条件下打开或关闭。 </li><li>在收购过程中，数据将自动保存。这些原始数据文件，然后通过Matlab软件脚本提供的三苯甲基自由基浓度和弛豫时间T 2的地图，这是翻译成通过现有的预校准的氧气浓度形象的图像处理。 </li></ol> 4。代表性的成果实验的结果是几个记录在不同的τ值的三维ESR微图像。图5典型的原始数据图像。前三名的图像，在黑暗条件下测得，除了在减少信号强度非常相似。另一方面，光线照射下的图像格局的变化，由于在不同部位的样品的不同的弛豫时间。此数据可处理1获得振幅图像，如图6所示和弛豫时间 T 2（图7）的图像。 T 2图像转化为通过一个预先存在的校准曲线，链接通过方程的弛豫时间的氧气浓度的氧浓度值： <img src="/files/ftp_upload/2122/2122eq.jpg" alt="公式1" />在这里，T 2 0探头的自旋弛豫时间在缺氧条件下（取决于探头的浓度，C和它的扩散系数D），k是比例常数。在大多数情况下，扩散系数不变化为生活样本（虽然，如果需要的话，它可以在原则上可以直接评估也由ESR 6，10），并在成像过程中获得的自旋浓度。因此，这种关系可用于直接测量氧气浓度。 回到图6，它是显而易见的，从幅度图像，蓝藻的细胞主要位于右侧的样品架。此外，根据图7，它是明确的，光启动生产的O 2，导致在该解决方案的O 2浓度显着增加，主要是在附近的蓝藻的体素。 <img src="/files/ftp_upload/2122/2122fig1.jpg" alt="图1" /> 图1：电子自旋共振的能量水平。 <img src="/files/ftp_upload/2122/2122fig2.jpg" alt="图2" /> 图2：典型的氧气浓度对荷瘤鼠的形象。左边的图像显示的解剖信息，根据MRI图像。一个稳定的有机自由基注入鼠标和ESR特性提供了在其环境中的氧气浓度（右）。 ESR为基础的结果MRI解剖图像上叠加。视场是32毫米。 <img src="/files/ftp_upload/2122/2122fig3.jpg" alt="图3" /> 图3：高分辨率的微观尺度ES的两个例子ř图像的光刻生成N @ C 60的粉末（左）和LIPC顺磁性晶体样品（右） <img src="/files/ftp_upload/2122/2122fig4.jpg" alt="图4" /> 图4：典型的哈恩成像脉冲序列显示微波（MW）和梯度，G X，G y和g ž脉冲。 <img src="/files/ftp_upload/2122/2122fig5.jpg" alt="图5" /> 图5：典型的原始数据ESR的微观图像：A，B和C为τ= 500600700 NS，分别测量没有光照的蓝藻样品的原始数据。项目D，E，并为A，B和C，但光照相同。强度是绘制在任意规模（但在每3个或深或浅的原始数据图像的设置一致） <img src="/files/ftp_upload/2122/2122fig6.jpg" alt="图6" /> 图6：相应的解决方案中的自由基浓度（任意缩放）幅图像。 <img src="/files/ftp_upload/2122/2122fig7.jpg" alt="图7" /> 图7：T 2图像和相应的[O 2]暗（左）和光（右）的条件下的值。

<ol>
	<li>Halevy, R., Tormyshev, V., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-imaging+of+Oxygen+Concentration+near+Live+Photosynthetic+Cells+by+Electron+Spin+Resonance.">Micro-imaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance.</a> Biophysical Journal. , (2010).</li><li>Blank, A., Suhovoy, E., Halevy, R., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESR+imaging+in+solid+phase+down+to+sub-micron+resolution:+methodology+and+applications.">ESR imaging in solid phase down to sub-micron resolution: methodology and applications.</a> J Phys Chem. 11, 6689-6699 (2009).</li><li>Gallez, B., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+EPR:+when,+how+and+why?.">In vivo EPR: when, how and why?.</a> NMR Biomed. 17, 223-225 (2004).</li><li>Eaton, G. R., Eaton, S. S., Ohno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EPR imaging and in vivo EPR. , (1991).</li><li>Matsumoto, 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+imaging+of+tumor+oxygenation+and+microvascular+permeability+using+Overhauser+enhanced+MRI.">Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.</a> Proc Natl Acad Sci USA. 106, 17898-17903 (2009).</li><li>Talmon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Porous+Media+by+PGSE.">Molecular Diffusion in Porous Media by PGSE.</a> ESR. J Phys Chem. , (2010).</li><li>Halevy, R., Talmon, Y., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+production+of+glass+sample+holders+for+improved+sensitivity+and+resolution+in+ESR+microscopy.">Photolithographic production of glass sample holders for improved sensitivity and resolution in ESR microscopy.</a> Applied Magnetic Resonance. 31, 591-598 (2007).</li><li>Allen, M. M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+Division+of+Some+Unicellular+Blue-Green+Algae.">Growth and Division of Some Unicellular Blue-Green Algae.</a> J Gen Microbiol. 51, 199-199 (1968).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> J Gen Microbiol. 111, 1-61 (1979).</li><li>Blank, A., Talmon, Y., Shklyar, M., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+diffusion+in+liquid+phase+by+electron+spin+resonance.">Direct measurement of diffusion in liquid phase by electron spin resonance.</a> Chem Phys Lett. 465, 147-152 (2008).</li></ol>

此协议表明如何ESR的显微成像可以应用到地图氧浓度附近居住的小样本。一个空间分辨率〜30 × 30 × 100微米证明，与附近的微摩尔的氧气浓度的敏感性和分飞摩尔每个体素的绝对氧敏感性。氧细胞附近的映射使用的ESR显微成像补充基于微电极或荧光/磷光目前可用的技术。此外，适当的顺磁探头，它会很容易地适用于细胞内的氧气显微成像，能力等方法发现很难实现。在不久的将来，我们计划进一步改进这种方法，提供活的样本图像，几微米的分辨率，提供对比参数，如超氧化物浓度，酸度（PH），探头的扩散系数，当然，氧气浓度。这些功能是相辅相成的对比类型和样本特征（非透明如厚样品，并在某些情况下，细胞内与外的测量）到目前的光学方法。

<table><tbody><tr><td>Centrifuge </td><td>Kendro</td><td>Heraus, 75003235</td><td></td></tr><tr><td>Perdeuterated triarylmethyl (trityl) radical</td><td>Novosibirsk</td><td></td><td>Synthesized at Novosibirsk using the method described in reference &lt;sup&gt;6&lt;/sup&gt;.</td></tr><tr><td>BG-11 buffer</td><td></td><td></td><td>For instruction preparation, see Scheme 1 and references 8, 9.</td></tr><tr><td>Syringe</td><td>Hamilton Co</td><td>Microliter 7000.5</td><td></td></tr><tr><td>Ultraviolet Curing</td><td>Norland Products, Inc.</td><td>NOA63, or NOA61.</td><td></td></tr></tbody></table>

electron spin resonance micro-imaging of live species for oxygen mapping

这个协议描述三维映射与微米级的分辨率1活细胞的直接环境中的氧含量的电子自旋共振（ESR）显微成像方法。氧气是在生命周期中最重要的分子之一。它作为在线粒体氧化磷酸化的终端电子受体和用于生产活性氧。氧气测量是很重要的线粒体和代谢功能，信号转导通路，各种刺激的影响，细胞膜的通透性，和疾病的分化的研究。耗氧量，因此，细胞的新陈代谢，这是广泛适用于各种生物系统对整个生物体的细胞线粒体的信息标记。由于其重要性，有很多方法被开发为氧气的测量实时系统。主要是根据当前的尝试，提供高分辨率的氧成像光学荧光和磷光方法，未能提供令人满意的结果，因为他们采用高光毒性和含氧量低灵敏度的探测器。血沉增快，措施，从外源性样品中顺磁探头的信号，被称为氧气的浓度，以提供非常精确的测量。在一个典型的案例，ESR测量地图探头的线型扩大和/或松弛时间缩短，直接链接到当地的氧气浓度。 （氧是顺磁性，因此，外生顺磁探头碰撞时，急促的弛豫时间。）传统上，这些类型的实验与分辨率低，毫米波规模小动物成像的ESR。在这里，我们将展示如何ESR成像还可以进行微米级的小活标本的检查。 ESR的显微成像是一个相对较新的方法，使收购接近室温 2 1微米的分辨率与空间分辨ESR信号。本议定书的文件的主要目的是为了表明，这种新方法，随着新开发的氧敏感探头，可以适用于小活样品中的氧含量的映射。一个空间分辨率〜30 × 30 × 100微米证明，与附近的微摩尔的氧气浓度的敏感性和分飞摩尔每个体素的绝对氧敏感性。氧细胞附近的映射使用的ESR显微成像补充基于微电极或荧光/磷光目前可用的技术。此外，适当的顺磁探头，它也将很容易地适用于细胞内的氧气显微成像，能力等方法发现很难实现。

Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

这个协议描述为微米级的三维成像在活细胞的电子自旋共振显微镜直接环境氧浓度的方法。

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

electron-spin-resonance-micro-imaging-live-species-for-oxygen

Research

JoVE Journal

Biology

22.3K 次观看.  The Technion, Israel Institute of Technology. 这个协议描述为微米级的三维成像在活细胞的电子自旋共振显微镜直接环境氧浓度的方法。

视频: Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

电子顺磁共振在生物样品环境温度和 77 k 的应用

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

科研

生物化学

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (&#181;s) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

使用基于频率域的摄像机系统的 O2的发光寿命成像

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

化学

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

生物工程

<meta charset="utf8"></head><body>使用 EPRI 和超声对肿瘤氧水平进行临床前成像

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

<meta charset="utf8"></head><body>肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像 

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

癌症研究

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

生物学

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

电子自旋共振氧测绘活体物种显微成像

摘要

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此视频中的章节

电子顺磁共振在生物样品环境温度和 77 k 的应用

使用基于频率域的摄像机系统的 O₂的发光寿命成像

快速扫描电子顺磁共振打开新的途径成像生理重要参数

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

经济实惠的氧气显微镜辅助多细胞球体生物制造

体内使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

电子自旋共振氧测绘活体物种显微成像

摘要

探索更多视频

此视频中的章节

电子顺磁共振在生物样品环境温度和 77 k 的应用

使用基于频率域的摄像机系统的 O2的发光寿命成像

快速扫描电子顺磁共振打开新的途径成像生理重要参数

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

经济实惠的氧气显微镜辅助多细胞球体生物制造

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

使用基于频率域的摄像机系统的 O₂的发光寿命成像

体内使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像