"在真核细胞中，利用生物信息学和分子生物学方法的建模细菌蛋白质异戊烯"玛丽·路易丝·安德鲁斯奖的癌症研究计划，弗吉尼亚州科学院主办，"血浆中的RNA编辑的生理意义的项目过程中，DIY低温检索设备已测试树突状细胞"由托马斯·Jeffress和凯特·米勒Jeffress纪念信托和国家合同16.740.11.0364（科教部，俄罗斯）。我们想感谢援助与RNA的完整性实验贝丝EOM。

1。大会Cryotolerant设备Cryoboxes检索<ol><li>用钳子，理顺3片面强领带（ 图1A），使L形图1b所示的强领带注:侧。强领带的尺寸可以选择取决于杜瓦颈部直径。 </li><li>确保两个这样的强强烈的领带ţ表带使用皇冠螺栓螺母，垫圈和螺钉形成Cryoscoop基地，在图3（AB）所示（ 图2）注:两强关系是用来允许检索5年半英寸x 5½英寸cryoboxes。 </li><li>使用开槽板（ 图4），强ţ领带皮带上的插槽与插槽对齐开槽板的一端，并确保与帮助希尔曼螺母和螺栓（ 图5），两项合计注:如果开槽板和强烈的领带并没有合作我与预钻孔，您将不得不使用硬质合金钻头孔钻。 </li><li>弯曲的钳子的帮助下，开槽板的另一端， 如图6所示。 （这一步是可选的）。 </li><li>包装用泡沫手柄，更好地掌握设备时，在液氮中， 如图6所示。 （可选） 注意:以高压灭菌前取出泡沫。 </li></ol> 2。大会Cryotolerant设备冷冻管检索 Cryotolerant DIY的检索设备，也可以适应冷冻管检索。这涉及到代不锈钢滤网平底舀出液氮瓶（ 图7）改变。 <ol><li>拧官方螺栓和螺母，开槽板强领带ţ表带附加。 </li><li>开槽板前附加不锈钢滤网，轻轻弯曲的颈部;这将会使设备更容易操纵，如果过滤网唇，用钳子弯唇:用钳子帮助定位在一个角度基地类似于勺子（ 图8）注意过滤。 </li><li>随着开槽板的孔对准过滤器手柄上的洞，并确保滤网帮助希尔曼螺母和螺栓， 如图8所示的注意的:如果过滤不预钻孔，您将需要安排有硬质合金钻头钻孔。 </li></ol> 3。检索的杜瓦Cryoboxes和冷冻管<ol><li>开始，放在低温手套，并删除1个或2个冰柜从杜瓦架到允许的回旋余地。 </li><li>轻轻刮去沿杜瓦底部的直立Cryotolerant DIY检索装有强领带ţ表带基地（ 图5）设备和收集箱fallen个底部的杜瓦罐注:液氮沸腾。立即用温暖的对象接触，包绕在绝缘氮气体（Leidenfrost效果）的对象。冷却装置之前，检索样本，慢慢地浸入到杜瓦设备，并允许液氮冷却，然后再进行上述步骤的设备足够。 </li><li>直立拉设备，沿墙壁杜瓦罐舀出冷冻箱。 </li><li>抓斗从杜瓦底部检索框。 </li><li>检索冷冻管，轻轻刮去杜瓦的底部装有一个过滤器（ 图9）cryoscoop。 </li><li>类似cryobox检索，正直和抢拉设备在过滤器收集的小瓶。 </li></ol> 4。代表结果在基因表达研究中的一个主要挑战是，RNA的质量取决于储存条件冷冻组织标本，提取RNA。 图10显示了样品处理的总RNA的完整性的影响。来自同一组织源（人体脂肪）的总RNA受到不同的样品处理。实验之前，完整的总RNA的preparate显示清晰的28S和18S rRNA基因与28S rRNA的乐队乐队约两倍激烈的18S rRNA带（ 图10，泳道1）。这个比例为2:1（28S:18S）是一个迹象表明，RNA是完整的，而部分降解的RNA出现一抹黑，或者其28S 18S强度比从2:1 5出发。泳道3-4描绘了显着降解的RNA样品。这些RNA样品经反复局部defreezing从杜瓦（方法A）的短期检索，从而建模与长期贮存定期滗由于堕落瓶和盒检索的必要性（ 图10巷3-4 ）。然而，样本存储在杜瓦服务使用自助设备（方法B），因此，没有偏defreezing，包含完整的RNA（ 图10巷2号）。 <img alt="图1" src="/files/ftp_upload/3903/3903fig1.jpg" /> 图1。 A）该基地由两个3双面强领带的尺寸。每一个强烈的领带，包含一个大型预钻孔（实心箭头）和多个较小的孔，每个面（小箭头）。这提供了一个选项，以允许用螺母和螺栓紧固。还小的孔允许在样本检索液氮快速排水B）强烈的领带一边用钳子的帮助，使L形强领带拉直。 <img alt="图2" src="/files/ftp_upload/3903/3903fig2.jpg" /> 图2。强领带ţ表带的用于保护强联系在一起。像强领带，T字表带一体的大型预钻孔（实心箭头）和两个较小的孔（SMaL公司升箭头）上的每一个角落。这可以确保沿长度的T强关系 - 表带使用的螺母和螺栓。这也使T型带固定槽板。 <img alt="图3" src="/files/ftp_upload/3903/3903fig3.jpg" /> 图3。一）每个强领带系T型带的一个角落，沿其长度冠螺栓螺母，螺栓和垫圈。 乙）两强关系获得彼此相邻，以帮助检索5½英寸×5½英寸cryoboxes。小cryoboxes如2英寸x 2英寸，单强领带可以直接固定开槽板，没有一个T-表带。 <img alt="图4" src="/files/ftp_upload/3903/3903fig4.jpg" /> 图4。开槽平板作为手柄使用。开槽板的长度取决于杜瓦的深度。在这种情况下，使用了4英尺开槽板。开槽板的厚度是非常重要的小号自从开槽薄钢板（&lt;14计），使设备薄弱，同时检索充满杜瓦液氮样品。 <img alt="图5" src="/files/ftp_upload/3903/3903fig5.jpg" /> 图5。强关系和T带平底组成，然后开槽板的一端固定在。其余的T表带角落较大的孔对齐平板插槽，并用六角螺栓和螺母紧固。该装置可用于检索从杜瓦cryoboxes。 <img alt="图6" src="/files/ftp_upload/3903/3903fig6.jpg" /> 图6。开槽平板的一端用钳子弯曲，并用泡沫覆盖，以提供更好的抓地力，特别是深杜瓦瓶，在这种情况下所示。 <img alt="图7" src="/files/ftp_upload/3903/3903fig7.jpg" /> 图7。滤网，丝网和不锈钢拉手用于检索从杜瓦冷冻管。如果手柄没有提供一个插槽，钻硬质合金钻头钻一个洞，将需要。 <img alt="图8" src="/files/ftp_upload/3903/3903fig8.jpg" /> 图8。为了便于检索的冷冻管，过滤器一起用钳子杓形滤网的颈部弯曲。如果唇在这种情况下，提供了过滤器，钳子可以用来弯曲。这可确保滤网内没有得到杜瓦捕获。 <img alt="图9" src="/files/ftp_upload/3903/3903fig9.jpg" /> 图9。过滤器的一端固定在平板开槽六角螺母和螺栓。 <img alt="图10" src="/files/ftp_upload/3903/3903fig10.jpg" /> 图10。样品的总RNA的完整性受到不同的样品处理的条件。探讨如何处理C样品的影响RNA的质量onditions，一套来自同一来源的RNA样品受到两种方法的装卸。在使用自助设备的样品检索杜瓦，因此，没有偏defreezing包括RNA的存储方法A。方法B遭受杜瓦短期检索重复的部分defreezing存储在杜瓦的RNA，从而建模与长期存储，检索下跌样品定期调迁。总RNA的完整性，然后用凝胶电泳分析。泳道1:样品前存储杜瓦;巷2:从的杜瓦样品遭受的检索方法A; 3巷，4:样品从遭受的检索方法B杜瓦，M:100 bp的阶梯。

<ol>
	<li>Kittel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cryogenic Engineering. , 41 (1996).</li><li>Edeskuty, F. J., Walter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stewart Safety in the handling of cryogenic fluids. , (1996).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. , (2011).</li><li>Muyal, J., Muyal, V., Kaistha, B., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutation Research/Reviews in Mutation Research. 543, 217-234 (2003).</li><li>Lynch, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CELL,+TISSUE+AND+ORGAN+CULTURE+|+Storage+and+Cryopreservation.">CELL, TISSUE AND ORGAN CULTURE | Storage and Cryopreservation.</a> Encyclopedia of Rose Science. , 106-111 (2003).</li><li>Crawford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics Engineering. , (1998).</li><li>Gorenc, B., Tinyou, R., Syam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Steel designers' handbook. , (1996).</li><li>Harper, C. A., Petrie, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+materials+and+processes:+a+concise+encyclopedia.">Plastics materials and processes: a concise encyclopedia.</a> Wiley-Interscience. , (2003).</li></ol>

没有利益冲突的声明。

检索cryoboxes和冷冻管从杜瓦罐底部的最常用的方法之一是迁置到一个备用的容器中的液态氮和拉出样品留在烧瓶或漂浮在倒出的液氮。然而，这是一个不安全的做法，可能会导致低温烫伤或窒息，由于长时间暴露在液氮蒸汽2。其他常见的方式检索堕落的冷冻管涉及使用离心管钳3。然而，黑暗的空间内充满液氮罐的能见度有限，限制了其可操作性。 如上所述?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>希尔曼集团镀开槽平1-3/8×4英尺14计</td><td>当地的五金商店</td><td> N / A </td><td> 1个单位的要求</td></tr><tr><td>皇冠螺栓不锈钢＃6 - 32×3/8英寸扁圆头十字径机螺杆</td><td>当地的五金商店</td><td>型号＃42281 </td><td>需要4个单位</td></tr><tr><td>冠螺栓3/8英寸x 1-1/2英寸镀锌钢护舷垫圈</td><td>当地的五金商店</td><td>型号＃20122 </td><td>需要4个单位</td></tr><tr><td>冠螺栓锌镀＃8-32机螺杆螺帽</td><td>当地的五金商店</td><td>型号＃18512 </td><td>需要4个单位</td></tr><tr><td>辛普森强铁BC40Z 4英寸半基地In.x4 </td><td>本地硬件购物</td><td>型号＃BC40Z </td><td>需要2个单位</td></tr><tr><td>辛普森强铁6英寸x 6英寸ţ表带</td><td>当地的五金商店</td><td>型号:66T </td><td> 1个单位的要求</td></tr><tr><td>奥奈达6"滤网</td><td>本地杂货店</td><td>型号:7859W </td><td> 1个单位的要求</td></tr><tr><td>该的希尔曼集团镀锌六角螺母3/8-16 </td><td>当地的五金商店</td><td>型号＃:810509 </td><td>需要6个单位</td></tr><tr><td>该的希尔曼集团镀锌六角螺栓3/8- 16 </td><td>当地的五金商店</td><td></td><td>需要4个单位</td></tr><tr><td>绝缘泡沫</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr><tr><td>螺杆驱动程序</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr><tr><td>钳子</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr></tbody></table>

do-it-yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

液氮是无色，无嗅，极冷（-196℃）的液体在压力下保持。它通常被用来作为一个长期的生物材料，如血液，细胞和组织1,2长期储存的低温液体。液氮低温性质，而理想的样品保存，可引起接触活组织快速冷冻-被称为"低温烧伤"2，这可能会导致密切合作，参与从杜瓦瓶样品的存储和检索的人严重冻伤。此外，液氮蒸发，降低空气中的氧气浓度，并可能导致窒息，特别是在密闭空间2。 在实验室，生物样品往往存储在冷冻管或不锈钢架堆放在杜瓦罐1 cryoboxes。提供这些存储架，以防止打滑从机架盒与长轴成底部杜瓦在常规处理。然而，过于频繁，箱或珍贵样品小瓶溜出去和充满液氮罐的底部下沉。在这种情况下，样本可以不厌其烦地检索后液氮转移到一个备用的容器中或丢弃它。然后可以相对安全地从回收空杜瓦盒和瓶。然而，低温液态氮，其扩张速度的性质，使沉船样本检索的危险。它通常被推荐样本检索从未由一个人进行了安全处。另一种方法是使用市售的酷抓斗或钳拉出瓶3。在黑暗的充满液体的杜瓦瓶的能见度有限，但在其使用带来的一个主要的限制。 在这篇文章中，我们描述了一个Cryotolerant DIY的检索设备，这使得从杜瓦样本检索含有低温液体既安全建设第二容易。

Watch this Scientific Journal Video about Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks at JoVE.com

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

在这里，我们提出了成本低，耐用cryotolerant设备样品从充满液氮的杜瓦罐检索。易于施工和设备的模块化设计使得低温储罐的样本检索过程的安全和方便。

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

do-it-yourself-device-for-recovery-cryopreserved-samples-accidentally

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

25.2K 次观看.  George Mason University. 在这里，我们提出了成本低，耐用cryotolerant设备样品从充满液氮的杜瓦罐检索。易于施工和设备的模块化设计使得低温储罐的样本检索过程的安全和方便。

视频: Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

修改MicroSecure玻璃化:一种安全，简便，高效的冷冻程序人胚泡

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

科研

发育生物学

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

生物化学

Cryogenic Liquid Jets for High Repetition Rate Discovery Science

This protocol presents a detailed procedure for the operation of continuous, micron-sized cryogenic cylindrical and planar liquid jets. When operated as described here, the jet exhibits high laminarity and stability for centimeters. Successful operation of a cryogenic liquid jet in the Rayleigh regime requires a basic understanding of fluid dynamics and thermodynamics at cryogenic temperatures. Theoretical calculations and typical empirical values are provided as a guide to design a comparable system. This report identifies the importance of both cleanliness during cryogenic source assembly and stability of the cryogenic source temperature once liquefied. The system can be used for high repetition rate laser-driven proton acceleration, with an envisioned application in proton therapy. Other applications include laboratory astrophysics, materials science, and next-generation particle accelerators.

This protocol presents the operation and principles of micron-scale cylindrical and planar cryogenic liquid jets. Until now, this system has been used as a high repetition rate target in laser-plasma experiments. Anticipated cross-disciplinary applications range from laboratory astrophysics to material science, and eventually next-generation particle accelerators.

用于高重复率发现科学的低温液体射流

This protocol presents a detailed procedure for the operation of continuous, micron-sized cryogenic cylindrical and planar liquid jets. When operated as ...

工程学

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

生物学

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

低温结构照明微观数据收集来自低温保存细胞

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

<meta charset="utf8"></head><body>优化受威胁珊瑚物种的珊瑚精子冷冻保存

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

<meta charset="utf8"></head><body>作者聚焦:为全球珊瑚礁恢复开发高效的低温保存和生物样本库技术

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

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用于高重复率发现科学的低温液体射流

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

低温结构照明微观数据收集来自低温保存细胞

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程

用于珊瑚精子冷冻保存以支持生物样本库和水产养殖的半自动化工作流程