我们想感谢他们的贡献，在发展的DR汽车技术伊万Schie和塞巴斯蒂安Wachsmann Hog​​iu。泰勒周承认从劳伦斯 - 利弗莫尔国家实验室的劳伦斯学者奖励计划“的支持。托马斯Huser是通过赠款援助计划的美国心脏协会的支持表示感谢。部分由美国国家科学基金会的资助下，这项工作也得到了支持。生物光子学中心，美国国家科学基金会科学技术中心，是由加利福尼亚大学戴维斯分校管理，根据合作协议的PHY 0120999号。也承认，从授予数量UL1 RR024146下的UCD临床转化科学中心从国家研究资源（NCRR）中心的支持。

1。汽车和DR -汽车信号的生成为了产生汽车和DR -车信号的同时，三个可调和同步短脉冲激光源需要。 <ol><li>为了获得三个同步脉冲，我们用一个10瓦的激光（PicoTrain，HighQ激光公司）开始。这种激光有一个固定的波长为1064 nm，7 ps的一个固定的脉冲长度，和一个76 MHz的固定重复率。 </li><li>使用了一系列的半波片和偏振分光镜立方体光束分裂成三个部分。一个半波片与偏振分束的多维数据集相结合，使我们能够调整在梁的每个组件的发电量，而不改变其方向。通常情况下，两束调整〜4.5 W每个，第三束包含了剩下的1 W </li><li>两个高功率光束，然后分为两个独立的光学参量振荡器（OPO，AVANTE，APE有限公司，德国柏林）执导。利用OPO的差频转换成两个低能量光子的不同波长的高能量光子。通过控制温度要达到这种效果的水晶，所产生的光子的波长可以被控制在0.1纳米。倍频这些信号，现在与一个固定波长为1064 nm的激光可以被转化成一个可调谐的激光束，780纳米至910纳米之间的任何地方。具有相同的1064纳米源抽两个独立的参量振荡器中，我们得到两个独立的自动同步到我们原来的泵浦激光器的可调谐激光源。 </li><li>第三个（最低功率）从1064纳米的泵浦激光器的光束是针对各地的参量振荡器的结合，使所有三束（2 OPOS，从泵浦激光器的1）可重组后的分色镜。为了有效地重组产生连贯的拉曼信号光子脉冲必须在时间和空间的重叠。由于OPO包含环腔，让每一个激光脉冲穿过晶体多次，通过发送一个额外的距离，造成了相当大的延迟，相对于原来的泵束的光束参量振荡器的旅游。这个距离必须得到补偿与第三光束通过引入额外的镜子，迫使这束前往其他两个相同的距离，才使用分色镜重组。 </li><li>为了提供每束行程可调延时阶段，使用棱镜retroreflectors路径长度的微调，引入到每束路径。 </li><li>另一半波板和极化光束分离器的多维数据集被添加到每束，让我们每束功率调整，独立不影响参量振荡器的输入。 </li><li>分色镜，先结合起来，从两个参量振荡器和1064 nm激光束，然后梁。必须小心，以确保梁正是重叠（即他们共线，并有类似的分歧）。这可以通过比较从梁重叠接近（几厘米内）和远（约1米）的分色镜检查。 </li><li>使用脉冲激光器的优点是样品，每个脉冲可以有一个高的峰值能量，允许连贯的拉曼信号的有效代，同时仍然保持较低的平均强度。高的平均强度，可能会损坏样品。因此，为了以进一步控制而牺牲峰值能量的平均强度，在三个结合梁是发送通过一个电光调制器（Pockel的细胞，ConOptics）作为一个脉冲选择器，可让我们来调整的的脉冲的重复率运作到达我们的样品，因此平均强度。 </li><li>合并后的激光束，然后再加到一个倒置显微镜具有高NA目标。目标通常是一个60 X水已纠正色差跨我们OPO的可调谐范围和1064 nm激光束一个数值孔径（NA）的1.2的物镜。紧张的重点由高数值孔径物镜所产生的允许最高效的新一代微米尺度上的连贯的拉曼信号。 </li><li>合并后的光束扩展以过量的显微镜物镜背面端口。通过溢出显微镜物镜的背面，我们可以达到最佳的聚焦条件，因此，最好在我们的显微镜系统的空间分辨率。 </li><li>在我们的显微镜物镜安装在一个XYZ压电阶段，这使我们能够获得通过光栅扫描束在样品，类似的商业束扫描共聚焦显微镜图像。 </li></ol> 2。使用三个短脉冲激光器使用三个短脉冲激光在几款车的信号生产结果，从不同的Combinations的两束激光，以及三色车，并从所有三个激光器结合DR - FWM信号。 <ol><li>信号的波长可以躺在光谱彼此接近。当信号并拢，它可能很难分开，以便他们使用固定波长的带通滤波器和分色镜分析。出于这个原因，我们的信号传递到一个成像光谱仪（SpectraPro 2300i，阿克顿研究），也作为一种有效的单色空间分隔在不同波长的信号。 </li><li>光谱仪在降低位置内的一个电子驱动的翻转镜背照射深枯竭电荷耦合器件（CCD）摄像头，提供了在整个信号范围内的光谱信息和发送信号，使我们能够确定和优化各种连贯拉曼信号。 </li><li>要选择信号，与我们希望的形象，我们只需旋转光栅光谱仪内，使用供应商提供的控制和数据采集软件（WinSpec，普林斯顿仪器），中心的高峰上的CCD相机的兴趣，然后改变翻转镜的位置重定向到这个信号的单光子计数的雪崩光电二极管（APD）已附加到第二个出口港。 </li><li>物镜，然后光栅扫描和APD的上记录的信号是用来产生一个图像所显示的每个像素，通过数据采集软件（SymPhoTime，Picoquant有限责任公司，德国柏林）的光子计数率。 </li><li>我们重复这种成像过程，为每个需要连贯的拉曼信号，这使我们能够比较在处理后的信号。 </li></ol> 3。样品制备为了获得清晰，重现性好的图像，必须采取一定的照顾，在准备样品。 <ol><li>样品通常是准备〜150微米厚的玻片上。这些盖玻片是够薄，以便与1.2 NA目标，通常用于高分辨率成像。 </li><li>激光灯是通过小的透明物体衍射光学诱捕介质对象时可能发生的。使用高度集中，高峰值功率，短脉冲激光束，然后可以拖动的小细胞沿着或细菌，造成图像模糊或有污点的。为了避免这种情况也可能是必要的首次应用旋涂一层薄薄的聚- L -赖氨酸样品固定在玻璃盖玻片表面。 </li><li>对于玻璃底培养皿中培养细胞，使成像，而不需要从他们的细胞培养中生长的菜分离细胞，可以利用。 </li><li>对于在这方面的贡献示范，我们先存入一个固定甲醛 C. 线虫线虫在玻璃盖玻片。 </li><li>然后添加一个5米氘蠕虫的葡萄糖溶液20μL，液滴。氘的葡萄糖溶液提供了一个独特而强烈的拉曼的背景签名。 </li></ol> 4。样品分析为了妥善照顾的双共振增强效应的优势，拉曼光谱的共振拉曼物质必须是已知的。 <ol><li>一旦样品准备使用共焦拉曼显微镜的盖玻片，以获得相关的自发的拉曼光谱和物色合适的峰分析。 </li><li>要执行的DR -汽车我们确定通常与血脂有关的CH拉伸模式，和CD拉伸模式的光谱位置，氘葡萄糖，是2845厘米-1和2121厘米-1分别。 </li><li>相干拉曼散射之间的两束激光的频率差时取得的一个分子的振动频率相匹配。通过调整参量振荡器817纳米探针的2845 cm - 1的CH模式时的1064纳米的激光束，并结合其他OPO的调谐868纳米，与1064 nm激光束时， 将探头2121 cm - 1峰。 </li><li>光栅扫描样品，对车的显微镜，我们现在可以观察三个连贯的拉曼信号。一车信号探测的CH伸缩振动，汽车信号探测CD拉伸模式，和DR的车信号探测两种。 </li><li>我们选择每个峰在2.3节中所述，图像，如上所述。 </li></ol> 5。图像处理中的基于这三个图像提取额外的信息，现在需要一些相当简单的图像处理。 <ol><li>首先必须正常化的图像。在每个信号生成过程中所涉及的每个激光强度可以实现由会计理论正常化。在实践中，然而，这并不总是工作，主要是由于非均匀的光谱响应分色镜，检测器和光栅。 </li><li>正常化的一个实用的方法依赖于CD的共鸣，在纯血脂控制信号的地区，应没有贡献任何信号。同样，在纯葡萄糖溶液地区的CH共振不应作出贡献的任何信号。考虑到这一点，我们的DR汽车图像和汽车上的氘葡萄糖溶液以及地区以外的C.光盘共振获得的图像正常化线虫蠕虫病毒，展览没有CH共鸣。 </li><li>然后，我们确定一个地区内的蠕虫病毒在CH谐振车的形象，血脂丰富和正常化相应区域内的归DR汽车图像。对于这种方法能够正常工作，我们假设，深入这个区域内没有氘葡萄糖的存在。这是一个安全的假设，因为脂类是疏水性的，不会混在一起的解决方案。 </li><li>现在，减去从规范化的DR汽车图像归一CH谐振车的形象，我们剩下的只是放大CD谐振信号。 </li><li>同样减去从规范化的DR - FWM形象正常化的CD -谐振车形象，我们剩下的只是放大CH谐振信号。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2085/2085fig1.jpg" alt="图1" /> 图1：图的DR - CARS显微系统如上所述。 <img src="/files/ftp_upload/2085/2085fig2.jpg" alt="图2" /> 图2：A C.白光形象氘葡萄糖溶液中的线虫蠕虫准备在玻璃盖玻片和成像 。 <img src="/files/ftp_upload/2085/2085fig3.jpg" alt="图3" /> 图3：修改后的油酸（不饱和脂肪酸），其中包括一个炔修改（碳三键的碳组） 在 2845厘米-1和2100厘米炔共振强的CH 共振拉曼光谱-1都从指纹的地区（密集峰地区）以及隔离，使他们相干拉曼成像的理想标志物。 <img src="/files/ftp_upload/2085/2085fig4.jpg" alt="图4" /> 图4：典型的三个短脉冲激光在样品重叠时产生的相干拉曼信号频谱中的箭头指向（ES）负责每个代表信号能量图的过程。在这里显示的图中虚线箭头表示，光子从激光参量振荡器的和坚实的箭头指示产生的信号。坚实的水平线显示的拉曼振动的能量，并提供一个可视化表示，在DR汽车，混合相同的3个输入光子同步探测两个不同的拉曼振动。 <img src="/files/ftp_upload/2085/2085fig5.jpg" alt="图5" /> 图5：典型的从成像C.结果线虫蠕虫使用DR汽车和汽车的前行所使用的三个信号如图4所示形象氘葡萄糖溶液的一种蠕虫病毒。在第二行中的图像是适当的规范化和排在第三的区别图像减去的DR汽车图像的汽车图像制作。

<ol>
	<li>Cheng, J. X., Volkmer, A., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+and+experimental+characterization+of+coherent+anti-Stokes+Raman+scattering+microscopy.">Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy.</a> J Opt Soc Am B. 19 (9), (2002).</li><li>Tolles, W. M., Nibler, J. W., McDonald, J. R., Harvey, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+the+Theory+and+Application+of+Coherent+Anti-Stokes+Raman+Spectroscopy+(CARS).">A Review of the Theory and Application of Coherent Anti-Stokes Raman Spectroscopy (CARS).</a> Appl Spectrosc. , (1977).</li><li>Weeks, T., Schie, I. W., Wachsmann-Hogiu, S., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+generation+and+Raman-resonant+imaging+by+non-degenerate+four-wave+mixing+under+tight+focusing+conditions.">Signal generation and Raman-resonant imaging by non-degenerate four-wave mixing under tight focusing conditions.</a> J Biophoton. 3 (3), 169-175 (2010).</li><li>Weeks, T., Wachsmann-Hogiu, S., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+Microscopy+based+on+Doubly-Resonant+Four-Wave+Mixing+(DR-FWM).">Raman Microscopy based on Doubly-Resonant Four-Wave Mixing (DR-FWM).</a> Opt Express. 17 (19), 17044-17051 (2009).</li><li>Schie, I. W., Weeks, T., McNerney, G. P., Fore, S., Sampson, J. K., Wachsmann-Hogiu, S., Rutledge, J. C., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+forward+and+epi-CARS+microscopy+with+a+single+detector+by+time-correlated+single+photon+counting.">Simultaneous forward and epi-CARS microscopy with a single detector by time-correlated single photon counting.</a> Optics Express. 16 (3), 2168-2175 (2008).</li></ol>

拉曼光谱和拉曼成像是在生物科学领域的强大的新兴工具。目前，这是在体内和体外细胞代谢和在加工和贮存的脂质代谢紊乱的研究尤其如此。大部分生物大分子中含有大量的类似，主要是碳基分子键，使拉曼光谱，从细胞和有机体获得通常是由脂类，蛋白质，核酸，糖类的贡献卷积，等血脂相对容易隔离从这些复杂的光谱，因为他们的倾向，形成密集的水滴或双层，因为它们含有大量的脂肪CH键的扩展链。然而，我们有能力隔离的复杂的细胞环境内特定的蛋白质，氨基酸，核糖核酸或DNA非常有限。如果感兴趣的这些分子是目前唯一在μM浓度及以下，尤其是这样。在这里，探测能力弱拉曼共振，利用我们新推出的DR -车不同的成像技术提供了一个潜在的强大的方法，其化学微量分析和成像。诚然，在本议定书中最复杂的部分是激光系统的路线和同步。当从头开始，同步脉冲，即确保脉冲在时间上是重叠的，尽管他们采取不同的路径可以通过使用脉冲自相关便利。一旦空间和时间上的重叠是实现，汽车和DR -汽车信号应易被察觉。然而，第一次对准往往是原油，造成弱信号。调整这个系统的最佳做法是，最初产生的微弱信号，并然后轻轻调整沿每条路径的反射镜和调整使用的延迟阶段的时间重叠，以改善信号强度。虽然光谱仪/单色行为，作为一个非常有效的挡板，室内光线干净的结果可以实现经营与室内灯光系统关闭，窗帘或镜头油管，以尽量减少其他各种光源（如电脑显示器推出的背景，指示灯，指示灯等）。 我们特别设置利用单光子计数雪崩光电二极管（APD）探测器和时间相关单光子计数（TCSPC）硬件检测5。这使我们能够极其微弱信号检测与相对低噪音，但许多团体与可变增益优势进行类似的测量时，发现了光电倍增管（PMT）。光电倍增管的优势是，他们提供的可变增益，有一个更大的探测区域，可以简化探测器的对齐方式。此外，我们的设置利用压电阶段转化的目标，为了实现光束扫描。这样做的好处是，我们有能力返回到以前扫描的图像具有较高的精确度内的任何部位，并采取额外的测量，包括自发的拉曼光谱。其他研究小组已经成功地利用扫描镜组件，甚至整个共焦扫描单元，如奥林巴斯FluoView系统，它提供了更快的成像能力，但它能够精确地在图像中任意位置的限制。 调谐激光器相匹配的拉曼共振也是一个重要的步骤，可能需要一些优化。虽然可称为拉曼峰从DR汽车和汽车获得最大的谱峰强度并不一定对应自发拉曼峰的最大值。这是由于内在的四波混合导致非共振背景信号和汽车，歪曲车光谱相对自发的拉曼光谱产生的信号干扰。光谱的位置，可以计算的汽车信号的峰值，但一个更实际的做法是调整几个小跨预期的位置拉曼共振光谱步骤OPOS的。这个过程应该产生一个明确的最高。事实上，从DR - FWM最敏感的共振都必须进行调整这个最大值。 的DR车的方法的最后一个潜在的问题也可以讨论，即DR的车信号，将取决于一个均匀分布的拉曼放大积极分子。对于大多数的生物对象，这很可能是广泛的OH共振，从水，这是丰富，几乎无所不在。水，然而，排除疏水区域，一个单元格，利用水的共振放大脂质模式时，得到的信号失真，如脂滴的领先。在我们的例子中，我们已经使用了一个氘葡萄糖溶液，产生不易察觉和丰富我们的生物样品的信号。同样，氘的水或氘生物buffeRS，如D - HEPES，都可以使用。在我们的例子中，脂滴内的C.线虫蠕虫足够小，总是包含在我们的系统聚焦激光光斑的氘的葡萄糖溶液和脂质。然而，这不是一般的真实。一个特殊的例子，将脂肪细胞，细胞质内产生相当大的脂滴。这意味着，与DR汽车技术进行任何实验，需要精心的准备和控制的实验来验证的结果。

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<td>60X water immersion objective</td>
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<td>Inverted Microscope</td>
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<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>ConOptics</td>
<td>350-160</td>
<td>&nbsp;</td>
<tr>
<td>Picotrain pump Laser</td>
<td>&nbsp;</td>
<td>HighQ</td>
<td>IC-1064-10000</td>
<td>&nbsp;</td>
<tr>
<td>Optical Parametric Oscillator</td>
<td>&nbsp;</td>
<td>APE</td>
<td>Levante IR</td>
<td>&nbsp;</td>
<tr>
<td>1.5 Glass cover slips</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-102 25cm-1</td>
<td>&nbsp;</td>
<tr>
<td>Half-wave plates</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>AHWP05M-980</td>
<td>&nbsp;</td>
<tr>
<td>Polarizing Beam Splitter Cubes</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>PBS052 or PBS053</td>
<td>&nbsp;</td>
<tr>
<td>Spectrometer/Monochromator</td>
<td>&nbsp;</td>
<td>PI Acton</td>
<td>Spectra Pro 2300i</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera</td>
<td>&nbsp;</td>
<td>PI Acton</td>
<td>PIXIS: 100B</td>
<td>&nbsp;</td>
<tr>
<td>Avalanche Photo Diode</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>SPCM-AGR-14-12691</td>
<td>&nbsp;</td>
<tr>
<td>XYZ Piezo Stage</td>
<td>&nbsp;</td>
<td>Physik Instruments</td>
<td>P 733-2CL P 721.CDQ</td>
<td>This is a combination of an XY stage and a Z objective holder</td>
</tr>
<tr>
<td>Dichroic Mirrors</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>Ff01-720/SP-25 LPD01-633RS-25</td>
<td>These specific dichroics are not critical, any set with the appropriate transmission/reflection characteristics will be sufficient.</td>
</tr>
<tr>
<td>Dichroic Mirror</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>Z830rdc</td>
<td>To combine the different near-infrared laser beams</td>
</tr>
<tr>
<td>TCSPC board</td>
<td>&nbsp;</td>
<td>PicoQuant</td>
<td>Timeharp 200</td>
<td>&nbsp;</td>
<tr>
<td>Symphotime Imaging Software</td>
<td>&nbsp;</td>
<td>PicoQuant</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Matlab</td>
<td>&nbsp;</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

differential imaging of biological structures with doubly-resonant coherent anti-stokes raman scattering (cars)

相干拉曼成像技术已经看到在过去的十年活动急剧增加，因为他们的承诺，使无标记的光学成像与分子特异性高 1 。然而，这些技术的灵敏度，幅度比荧光较弱的许多订单，要求毫摩尔1,2分子浓度。在这里，我们描述了一个从强或丰富的拉曼散射获得其信号放大技术，可以使弱拉曼活性分子或低浓度的检测。短脉冲激光在生物样品的相互作用产生的各种连贯的拉曼散射信号，每个对样品进行了独特的化学信息。通常情况下，这些信号，如相干反斯托克斯拉曼散射（CARS），只有一个用于生成图像，而其他的被丢弃。然而，当这些信号，包括汽车三色和四波混频（FWM），收集和比较，汽车信号，否则就很难检测到的信息可以被提取3。例如，双共振车（DR车）是建设性的两个共振信号干扰的结果。我们将演示如何2845厘米-1 CH脂质的伸缩振动和2120厘米-1的CD伸展的一个氘分子（如氘代糖，脂肪酸等）的振动产生的DR -汽车信号所需的三个激光微调可以用来同时探头都拉曼共振。在这些条件下，除了汽车从每个共振，合并DR车信号探测无论是也产生信号。我们演示了如何检测的DR车的信号，并从丰富的分子的振动放大信号，可用于增强信号较弱的敏感性之间的差异。我们进一步证明，这种做法甚至延伸到应用两个信号产生不同的分子，例如，使用强溶剂的拉曼信号，这样可以提高稀溶质的拉曼信号弱。

Watch this Scientific Journal Video about Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS at JoVE.com

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

结合三个单一波长的短脉冲激光器是用来产生相干反斯托克斯拉曼散射（CARS）和双谐振车（DR车）。这些信号之间的区别，否则很难察觉连贯的拉曼信号增强的灵敏度，使弱拉曼散射成像。

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

differential-imaging-biological-structures-with-doubly-resonant

Research

JoVE Journal

Biology

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

13.6K 次观看.  University of California, Davis. 结合三个单一波长的短脉冲激光器是用来产生相干反斯托克斯拉曼散射（CARS）和双谐振车（DR车）。这些信号之间的区别，否则很难察觉连贯的拉曼信号增强的灵敏度，使弱拉曼散射成像。

视频: Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

科研

生物学

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

氧成像在生物系统中的磷光纳米探针的合成和校准

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

蓝宝石OPO基于激光的标准激光扫描显微镜：在TI相干反斯托克斯拉曼散射（CARS）系统的实现

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

黄荧光蛋白标记 p62 在攻击样诱导结构中光漂白后的荧光恢复

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory&#039;s High Flux Isotope Reactor

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

橡树岭国家实验室高通量同位素反应堆的中子射线照相和生物系统计算机断层扫描

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...