我们承认资金来自英国国家计量局的化学和生物计量程序。我们感谢塞巴斯蒂安面包车林德加德，米克洛什Erdelyi和埃里克·里斯的技术咨询和建议。我们感谢米克洛什Erdelyi，埃里克·里斯和最大Ryadnov的有益的意见和对这个稿件的讨论。

注：在本协议中，提示和建议被发现以下一些步骤。符号“* 1”表示音符1是相关的这一特定步骤，依此类推。 1。玻璃的荧光葡聚糖涂层<ol><li>加入200微升的0.01％聚-L-赖氨酸溶液至8室盖玻片的各孔中并孵育10分钟。用移液管除去，以确保没有液体是剩下的。 </li><li>根据制造商的说明来创建一个2毫克/毫升的储备溶液重组葡聚糖的Alexa 647 * 1。从此，稀0.25微升到25微升的去离子水以创建20μg/ ml的溶液。 </li><li>创建的葡聚糖的Alexa 647溶液的高密度涂层稀释20微升的20微克/毫升的溶液加入到总共200微升去离子水中。对于一个中等密度稀溶液2微升在200微升去离子水（从原来的储备溶液1:10,000稀释）。对于低密度溶液稀释0.2微升在200微升去离子水（从原始原液1:100稀释）。 </li><li>加入200μl稀释的葡聚糖的Alexa 647的解决方案到每个孔中并孵育10分钟。较长的孵育时间可用于这可能导致更致密的葡聚糖涂层。捞出洗净与H 2 O 3倍* 2 </li></ol> 注1：其他葡聚糖-染料结合物都可以使用。未缀合的葡聚糖也可以缀合的染料如果需要，组合是无市售。 注2：相同的右旋糖酐室可以重新镜像在一个星期内，虽然荧光的均匀性可能会降低。建议贮存于4℃在没有任何缓冲。 2。荧光肌丝玻璃<ol><li>加入200微升的0.01％聚-L-赖氨酸溶液到室的各孔中并孵育10分钟* 3。回复用移液管移动，以确保没有液体的剩余量。 </li><li>加90微升一般肌动蛋白缓冲液（根据制造商的说明配制）的各腔室中。加10微升的10μM预制肌动蛋白丝溶液和1μl的鬼笔环肽-的Alexa 647（0.2单位，溶于1.5毫升甲醇中，当按照制造商的说明），并轻轻地吹吸以混合。温育30分钟* 4。 </li></ol> 注3：增加腔导致更少的纤丝结合到聚-L-赖氨酸包被的玻璃内的灯丝溶液的体积。 注4：孵化多达48个小时的时间，在4℃下进行了测试，效果良好。肌动蛋白丝的图像质量劣化后的腔室已经暴露于切换缓冲器所以不推荐使用相同的样品为一天以上。 3。微球荧光珠作为基准标记<ol><li&gt;稀释1微升的TetraSpeck珠到200微升PBS混匀。 </li><li>加入稀释珠到成像室，并等待15分钟* 5。除去溶液，并用PBS成像之前洗涤3次。 </li></ol> 注5 -稀释和培养时间可以调整，以得到的珠粒密度不同。该协议通常会导致在3 - 10珠每20.5微米视场的X 20.5微米。 4。表皮生长因子细胞染色<ol><li>培养HeLa细胞在成像腔室，以在Dulbecco改进的Eagle培养基（DMEM）约80％汇合的补充有10％胎牛血清（FBS）和1％青霉素 - 链霉素溶液。 </li><li>除去培养基，洗涤用PBS。然后10分钟，加入500μl的4％多聚甲醛溶液（4mL 10％甲醛溶液，将1ml 10X PBS中，加入5ml水）。除去甲醛和洗3次，用PBS洗涤。不要让细胞保持ÐRY和总是用PBS缓冲解决方案，以避免低渗应激。 </li></ol>注意 - 甲醛是剧毒。确保适当的手套，护目镜和实验室外套的磨损。检查处理甲醛本地的指引。 <ol start="3"><li>根据制造商的说明来创建50微克/毫升原液重组表皮生长因子的Alexa 647。加入2μl此储备溶液以200微升的0.1％BSA溶液（在PBS中）。从细胞中除去PBS，加入EGF溶液并孵育30分钟。 </li><li>去除溶液，并加入0.1％BSA（在PBS中）的15分钟时，封闭液之前，用PBS洗3次。除去这一点，并用PBS之前，成像洗再进行3次。 </li></ol> 5。交换缓冲液制备<ol><li>使酶原液（A）与10微升过氧化氢酶（20微克/毫升），20微升的1M Tris（2 - carboxyelthyl）膦盐酸盐（4毫摩尔）2.5毫升甘油（50％），125微升含1M KCl（25毫摩尔），加入100μl的1M pH为7.5的Tris-HCl（20毫摩尔），5毫克葡萄糖氧化酶（1毫克/毫升），并补足至5ml体积与diH20。这足以使100×50微升等分试样，它可以储存在-20℃，并使用长达1年。 </li><li> ）使葡萄糖储备液（B）与4克葡萄糖（100毫克/毫升），加入4ml甘油（10％）和36毫升diH20。这足以使100×400微升等分试样，它可以储存在 - 20℃，并使用长达1年。 </li><li>使还原剂原液（C）和113.6毫克MEA-盐酸（1M）和1毫升DIH 2 0。使用新鲜的或使10×100μl的等分试样，它可以储存在-20℃，并用长达1个月（不要重新冷冻等分试样）。 </li><li>只是之前成像，混合酶（A），葡萄糖（B）和还原剂（三）储备液一起在比50微升，400微升，100微升和弥补到1毫升的PBS * 6。这个缓冲器现在将清除氧并具有还原性的环境（100mM的MEA-HCl中）* 7。最终的pH值应在范围6.0 - 8.5（调整通常不是必需的）* 8。 </li></ol> 注6 -此缓冲区是适于与羰花青类染料等的Cy5和Alexa 647的使用。 注7 -最终的酶浓度的葡萄糖氧化酶的50微克/毫升（5单位/ ml）和过氧化氢 ​​酶的1微克/毫升（40-60单位/ ml）。酶活性（单位）是由供应商所提供和可以变化。在计算过氧化氢 ​​酶假设1微克/毫升终浓度为5.4的步骤后，将含有50单位的酶。各种缓冲组合物，包括类似的氧清除组件，可以发现21,30,40。对于缓冲液和染料组合的摘要见参考文献28,29。甘油和TCEP需长期贮存于-20℃。 注8 -如果成像微丝加2毫米MgCl 2和0.2毫摩尔ATP，以帮助稳定吨他细丝。 6。显微镜收购暴风影音资料（使用Alexa的647染料） <ol><li>接通STORM / PALM显微镜，成像之前优选至少3小时，以允许热平衡达到。在此之前成像具有漂移的机会增加。 </li><li>插入成像室到显微镜载物台样品架。确保样品是牢牢的持有人，是平的。 </li><li>从成像室中取出PBS中，然后填充到与开关缓冲器的顶部。小心地将盖玻片在室的顶部，确保没有气泡在所有和整个腔室被覆盖。补足额外的缓冲液或PBS如有气泡，否则视为缓冲性能可能会降低。 </li></ol>注意 - 以减少相对于所述物镜的样品的侧向和轴向漂移的可能性，建议在样品和缓冲液都留给平衡至室温至少15分钟前成像。 <ol start="4"><li>找到感兴趣的荧光结构进行成像。选择照明的期望的角度，在这种情况下，全内反射荧光或高度倾斜的角度被推荐为这种改善了信号 - 噪声比通过减少失焦的光，这是特别有利于对细胞样品。采取结构之前，暴风影音成像参考衍射极限的形象。 </li><li>增加激发激光（具有约640纳米的合适的激发波长下）到一个高功率，对应于约2千瓦/厘米2，而成像用的10毫秒曝光相机设定和大约50赫兹的周期时间（帧每秒） 。一旦荧光团已过渡到稀疏闪烁模式（大概在10秒内对大多数样品）采集10,000帧* 9。 </li></ol> 注9 - 10,000帧适合于这里描述的样本但它可能有必要增加帧编号，以50,000或更多的其他样品。 7。从原始数据中重建超分辨率图像（使用暴雨） <ol><li>在MATLAB中打开rainSTORM.m文件并运行（ 图1A）。 </li><li>在暴雨窗口（ 图1B）选择要使用的浏览按钮进行分析的图像文件。这应该是一个。tif文件（ImageJ的，可用于转换其它文件类型转换为TIF格式）。改变像素的宽度相匹配用于采集数据* 10显微镜系统的原始数据。保留其他值设置为默认。 </li></ol> 注10 -典型的EMCCD相机具有16微米的像素尺寸，以便用100X物镜系统上的图像上的像素的大小将是160纳米。对商业系统的像素大小是在软件中通常显示。像素大小100纳米和160纳米之间各有不同，最本土化的显微镜。<ol start="3"><li>按“图像处理”按钮，并等待一个初步的形象出现。该处理的持续时间将取决于文件大小，计算机说明书和原始数据* 11内的候选眨眼的次数。 </li></ol> 注11 -肌动蛋白和EGF数据10,000帧序列，花了23秒和25秒使用电脑与英特尔®至强®E5420 CPU分别处理。使用相同的计算机，而无需在MATLAB并行处理工具箱，或与电脑没有多核心处理，时间分别为66秒和81秒分别。 <ol start="4"><li>要生成一个更新的超分辨率图像按“打开审阅”按钮，会出现第二个窗口（ 图1C）。按以随意（ 图1D）来改变图像显示的“调整对比度”按钮。在某些情况下，当图像很暗的最大值应被减小。 </li><li>使用转iewer窗口（ 图1C），以产生有用的图像质量度量和改进的超分辨率图像进一步。将前三个质量控制参数4000，0.1和0.8〜2.0的值分别为* 12。更新每个光子的价值，它要么是基于光子校准测量，或使用由相机制造商特定的EM增益设置指定光子曲线校准曲线值的计数。这是为获得准确的精度和分辨率的测量* 13的关键。 </li></ol> 注12 -信号计数拒绝基于亮度的标准闪烁。该值越高越亮闪烁必须成为接受为最终图像本地化。这可能需要取决于染料和曝光时间和照相机的感光度的光子输出被改变。公差拒绝闪烁是否存在的信号，并拟合高斯例如，d之间显著方误差OUBLE峰。 PSF西格玛范围拒绝闪烁如果PSF的配合宽度超出此范围，即范围较窄，可用于拒绝了焦信号和不断荧光大汇总斑点。使用范围更广泛，可能会导致出现在最终图像错误定位工件。在实际应用中，建议使用定位精度的截止参数进行初步的质量控制。 注13 -更多的分辨率指标见参考文献23,27。在简要通过了解由每个所产生的光子数闪烁，可以计算出每个定位的定位精度，从而导出经整体的意思是精确估算整个图像。使用安道尔IXON DU-897E与200元价值的光子计数的增益设置为9.04。 <ol start="6"><li>改变定位精度截断（ 图1C）平均优化本地化PRECIS之间折衷离子对最终图像的定位号码。 30-50 nm的值取决于数据的质量和样品推荐。无论是直方图和info.txt文件的文件可以被用来告知决定（ 图1F和1G） </li><li>重构的比例因子（ 图1C）缺省值是5，这将产生32纳米的超分辨率像素假设在原始图像中的像素的宽度为160nm。如果原始图像具有100nm的像素尺寸，这将产生具有20nm的像素的超分辨率图像。增加比例因子在最终图像* 14，产生更小的超分辨率像素。 </li></ol> 注14 -定位显微镜的分辨率取决于所需的可视化（即一个简单的柱状图重建的像素大小），以及定位精度的平滑。在实践中，重建的像素尺寸一般应比定位小精度（见平均精确度估计度量中的信息文本文件 - 步骤6.11和6.12）。在这种情况下解决因重建像素大小的损失会很小23,27。例如，行和图4E的列方向的平均精度估计是16.1 nm和16.2 nm的。为16nm的像素大小已经被用于可视化这个数据（共10个重建的比例因子为160纳米的原始像素的宽度）。 <ol start="8"><li>修改限制帧的范围（ 图1C），以重建限制到的原始数据的子集，例如[2001-INF]将排除原始数据的初始2000帧。 </li><li>按&#39;运行评论家“按钮（ 图1C），以生成更新的超分辨率图像（ 图1E）。 </li><li>按“查看直方图”按钮（ 图1C），为了显示图像质量指标（ 图1F）* 15。 </li></ol&gt; 注15 -在右上角描绘了对摄像头的帧数，并在右下角（ 图1F）汤普森本地化精密工业直方图接受本地化的数量的曲线图可以用来告知定位精度截止审查选项。例如，使用50纳米的截止将排除所有与被包括在最终的超分辨率图像恶化精度的本地化。 <ol start="11"><li>按“保存图片”按钮，在评审（ 图1C），以保存所有这些数据* 16。 </li></ol> 注16 - 3至5个文件可以根据选定的（总和形象，STORMdataImage，STORMprocessed，信息及hists）的选项被保存， 见图1G。风暴处理后的图像会显示修改对比度和色彩映射。该STORMdataImage是灰度图像，其中每个像素的灰度级等于麻木呃这是在它确定本地化的。 <ol start="12"><li>检查数据后（ 如 info.txt文件和柱状图）返回到步骤7.6，如果需要用不同的审稿参数重复后续步骤。按“保存图像”在步骤7.11将覆盖以前保存的数据。 </li></ol> 8。箱跟踪和漂移校正（使用暴雨） <ol><li>按照步骤7.1 - 7.9以生成图像以正常的方式。 </li><li>按在审查员（ 图1C）的“框跟踪”按钮，然后单击并拖动一个框的审查图像上感兴趣的基准标记或结构。 </li><li>等到一个新的形象出现，这将显示“盒装位置”（ 见图6B，6C和6F的例子）。如果需要保存该图像。 </li><li>如果感兴趣的对象是一个基准标记，然后漂移校正，可以通过点击“设置锚”键，接着按&#39;子进行道漂移“按钮。最后单击“运行检阅者再次产生一个新的暴风影音图像，现在将所有本地化根据基准标记位置的校正。珠位置从最后的审阅图像删除。 </li><li>如果有最终的漂移校正后的图像中的其他基准标记，这些都可以通过按&#39;盒跟踪“被删除，”删除盒装&#39;然后根据需要“运行检阅者多次。 </li></ol>

我们什么都没有透露。

我们显示了一些简单的测试样品和可自由可用的处理软件暴雨能够优化STORM超分辨率成像。这些工具和方法将提供有用的起点为新用户和途径有经验的用户，以增加信心，他们的显微镜及其使用它们来研究生物学和细胞结构以前所未有的细节。通过使用具有已知的或容易理解的结构和算法，可以产生许多有用的图像质量度量，例如平均的估计精度，定位数和直方图表示有可能提高图像质量，并有更大的​​信心样本的组合在风暴成像过程。有没有一个放之四海而皆准的方法来采集数据，因为有许多不同的商业和自建显微镜配置，都可以使用。此外，有许多样品制备和图像采集步骤，它可以十字形ENCE因此最终图像有软件工具和测试样本是理解和优化暴风影音图像质量的关键。 另外，这些协议可以提供故障排除任何可能出现的问题，有用的和更加明确的方式。例如葡聚糖样品，以确定是否有任何的激光束对准或样品的照度不均匀一个非常有用的方法。如果有顾虑，开关缓冲区可能无法正常工作非常简单直观的测试，看看是否有任何&#39;闪烁&#39;会有所帮助。肌动蛋白丝是采用半高宽比较，以及突出的漂移和错误定位工件测量分辨率的有效途径。然而，应该指出的是，作为定位基础的超分辨率的方法可以是荧光团密度敏感，尽管其他因素，如曝光时间，帧速率，激光功率和图像处理方式，它是不可能分配一个分辨率为特别是显微镜和假设所有的图像都会有该决议。这是可能的，但是，是测量分辨率的各个方面，例如平均定位精度，定位数和漂移27。即使基准标记不能被纳入每一个实验，他们可以，至少，可以用来表征显微镜系统，它的环境和更好地了解运作方面的考虑，如多少时间才能达到热平衡启动后。以及校正横向漂移，基准标记可用于表征和正确的轴向漂移，虽然这要求使用一个象散透镜和反馈机制来纠正样本位置而被获取的图像43。商业超分辨率系统通常包括某种形式的稳定控制或聚焦锁定机构，其可以是用于校正焦点偏移的更实际的解决方案。 有再替代非特异性镀膜玻璃用葡聚糖，如直接使用染料或其它共轭分子，如第二抗体。这些方法的局限在于不知道分子结合于玻璃的数量。已经使用的另一种纤维结构，包括微管28和21的DNA。然而，非常小的尺寸和方便的市售试剂的结合，使肌动蛋白的有吸引力的替代，特别是发散或支长丝的间隔距离也可以是系统性能27的有用的测量。 有余地测试样品的进一步发展，尽管在这一领域28,29,46,47最近进展。特别是，多彩色成像提出了许多挑战在成像的全部3个主要方面，样品标记，图像采集（硬件）和软件（图像处理和对准）。有一个被一些出版物寻址这些方面4-6，因此很清楚，合适的测试样品和方法是对生成并可靠地解释这些类型的图像的关键。事实上，最近它表明，dSTORM可以用来采取两种彩色图像，并解决，核孔复合体的高度对称的性质，作者认为，这将是评估的色差校正45性能的理想方法。另一个有趣的方法是利用DNA折纸的创建nanoruler，这在特定的子衍射的距离相隔46创建定位的荧光团的可能性。这提供了评估分辨率，使测量距离的一种方式。然而，最终的目的是将这些超分辨率技术适用于非理想化的样品，如细胞，这是复杂的三维结构。在这种情况下，更透彻的了解吨的组合他技术结合软件工具，帮助用户在获取数据，以适当的方式处理它，并提供图像指标很可能是最终的答案。28,29,47,48

最近已经开发出了一系列光学显微技术，通常被称为超高分辨率显微镜或纳米显微，这可绕过衍射极限，并具有100nm或更1,2分辨率生成图像。由于超分辨率显微镜公布，因为这是一年中自然 - 方法学方法在2008年，出现了大量的本土化为基础的显微镜发展。例如，有多种颜色的应用程序3-6，3D实现7-12，甚至活细胞的应用5,13-17。此外，由于这些系统都被商业化，现在使他们的出路光学实验室进入细胞生物学家手中有可能是生物医学问题进一步增加他们的使用和应用。 该家族中的一个分支是通过建立一个图像分子通过分子工作的定位为基础的方法。我n阶做到这一点，大部分样品中的荧光分子必须被关闭，因此，只有少数的空间上分离的分子是活性在任何一个时间。这些分子然后被迅速接通和断开与许多图像的拍摄，使人口的显著部分成象，以反映下层结构与子衍射精度。许多方法已经公布，包括GSDIM 18，PALM 19，FPALM 20，STORM 21和RESOLFT 22。用于测量一个单分子检测的位置的算法可以被用于定位该分子的实际位置与精度比使用高斯拟合23 20毫微米较好，例如rapidSTORM 24,25 QuickPALM 26 palm3d 12和暴雨27。当成像细胞或其它生物样品，其具有高的分子密度，因此，有必要采取几千帧的这个闪烁，在空间上分离，信号以图像的标记的分子的显著比例。 随机光学重构显微术（STORM）和高度相关dSTORM和GSDIM方法是定位为基础的超分辨率的方法，这已被证明与市售的有机染料21的工作。此后，他们被证明是适用于许多不同的染料28-30，这使得由于特异性和完善的免疫标记的方法来进行荧光显微镜相对方便的方法特别有吸引力。因此，人们很容易获得各种染料抗体和染料生物分子结合物，它在本地化为基础的超分辨率成像中使用的潜力。虽然暴风影音和类似方法的一般原理也比较简单，和一见钟情的硬件和样品制备看起来比较STRAightforward，有许多的过程步骤，这是实现高品质，无瑕疵的，超分辨率图像的关键。 如同所有的显微镜技术的方法可以在3个主要步骤被认为是：首先，样品制备，第二，图像采集和第三，图像处理和/或图像分析和解释。额外的解析力和产生超分辨率图像使用本地化为基础的方法的方法，引入了一些新的问题和注意事项31-33。与样品制备开始，进行免疫标记，尤其是间接免疫荧光法，其中初级和次级抗体组合使用时，应该指出的是，荧光染料可高达20nm的远离感兴趣的分子。在微管的情况下，这将导致在直径为35-50纳米，具有25纳米28,30的预期的微管的直径相比较。此外，该标记密度应该米EET奈奎斯特准则，以便产生高质量的图象14。例如为20纳米沿丝状结构的预期的图像分辨率应该有一个染料分子至少每10纳米27。虽然许多染料已发表的工作进行本地化的基于接近染料的整体性能是至少四个重要的准则，每个开关事件，即检测到的光子（各眨眼的亮度 - 亮越好）的混合，对平衡 - 断电占空比（染料在关闭与打开状态的对比度 - 更多关一般是越多越好），存活分数（低白化率越好）和开关周期数（每荧光闪烁的数字 - 更多对于高解析度好，虽然每荧光闪烁1次可以为分子计算目的的优势）。这种情况是由一个事实，即这些属性对于任何给定的染料可以以不同的缓冲条件而变化，并进一步复杂与激光功率28,29。此外，还有这些独特STORM考虑，图像质量可以通过在通过最小化由自发荧光修饰的固定条件和使用的猝灭试剂相同的方式更为传统的荧光显微镜技术，例如优化的样品制备改善。此外，最小化非特异性结合的荧光基团是很重要的，这可以通过调整标签的浓度，孵育时间和阻断和洗涤步骤来完成。 图像采集的第二个步骤也很关键。在显微镜上的最佳设置将取决于染料，缓冲液条件，标签密度和样品类型， 例如单分子膜在玻璃上，将细胞或组织样品。主要的考虑因素是相机的曝光时间，帧号，照射角度（TIRF，希洛，EPI）和激光功率。其目的是尽可能快地收集尽可能多的明亮空间上分离闪烁。如果我的背景的甚高或闪烁不很明亮，换句话说，信号与噪声的比率差，重建算法将无法向的位置作为精确定位。以及最大限度地提高定位的精度， 即与每个分子位置的准确性可以被测量，图像质量也依赖于大量的本地化的。如果感兴趣的分子的数量不足进行成像，无论是由于不良的标记效率，过度漂白或不足的帧数，然后将所得的超分辨率图像将点状和不连续的奈奎斯特准则将不会得到满足14,27 。 最后，用标准荧光显微技术相比，在第三步骤中，图像处理，就显得尤为重要。有一个广泛的自由和市售的算法，这既是优点，在选择方面，也是缺点，因为它可以混淆知道瓦特HICH是最适合使用。例如，如果原始数据有良好的间距空间上不同的闪烁然后单分子拟合算法可以非常准确，高效，例如通过在暴雨27可疏拟合算法，rapidSTORM 24,25，palm3d 12和QuickPALM 26软件。如果原始数据中含有大量的重叠信号，然后其可以是更合适的算法包括DAOSTORM 34 3B 35和deconSTORM 36。此外，这些算法包含的阈值不同的标准，如信号计数，或者每闪烁光子，以及各种不同的图像显示选项，例如具有平滑的高斯函数的可视化或作为直方图与像素网格，其中所述超分辨率像素大小可以是由用户选择。所有这些选项更改最终图像的外观和对图像判读和分析的影响。 <p class="jove_content"&gt;因此，我们提出了3个试验样品，将提供新的用户提供了一个起点，基于染料的定位为基础的超分辨率的实验，其中，为了清楚起见，我们将称之为STORM，有经验的用户有机会进一步优化他们的实验。首先，有可能以涂覆玻璃带的葡聚糖 - 染料结合物的荧光层的表面上。这提供了一个快速和简单的方法来建立的显微镜，染料和缓冲正努力产生闪烁的荧光信号。该方法可以再通过比较由暴雨产生的平均定位精度和数量指标来优化缓冲条件下，图像采集的设置和测试不同的染料进行扩展。其次，我们提出了一个简单的协议，用于在玻璃上，可以使用毒伞素染料结合物很容易准备标记肌动蛋白丝。这些提供了理想的结构，采取分辨率27和通常的半峰全宽（FWHM）的测量的灯丝是作为第37的经验估计。但应注意的是，分辨率样品之间和同一样品内的图像之间变化，因为在定位基于超分辨率显微镜的分辨率是荧光的亮度，背景噪声和本地化密度27的功能，并且这些不一定恒定整个样品。肌动蛋白丝，是要证明潜在的文物，如mislocalizations和漂移，以及他们如何可以在暴雨的软件功能来识别。此外，我们描述了使用的荧光基准标记到两个测量和正确的漂移。第三，细胞与表皮生长因子（EGF） - 染料偶联物的染色进行说明。 EGF提供的超分辨率图像的性能的有效指标，因为该受体在细胞表面的比例通过囊泡，它们是约50〜150 n个子衍射尺寸的结构经历胞吞作用米直径27,38,39。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Labtek Imaging chamberglass</td>
			<td>Fisher</td>
			<td>CBR-166-390E</td>
			<td>Sample preparation</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>* See below for alternatives</td>
		</tr>
		<tr>
			<td>100 X TIRF Objective Lens</td>
			<td>Olympus</td>
			<td>UAPON 100XOTIRF</td>
			<td>* See below for alternatives</td>
		</tr>
		<tr>
			<td>EMCCD Camera</td>
			<td>Andor</td>
			<td>iXon 897</td>
			<td>* See below for alternatives</td>
		</tr>
		<tr>
			<td>640 nm laser</td>
			<td>Toptica</td>
			<td>iBEAM-SMART-640-S</td>
			<td>*150 mW - for imaging Alexa 647 or Cy5 dyes</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments</td>
			<td>&nbsp;</td>
			<td>*Acquisition software (controlling hardware)</td>
		</tr>
		<tr>
			<td>PC</td>
			<td>Dell</td>
			<td>&nbsp;</td>
			<td>Windows XP (64 bit), Intel Xeon E5420 CPU, 16 GB RAM *Instrument control and image processing</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Image analysis and processing <a href="http://rsbweb.nih.gov/ij" target="_blank">http://rsbweb.nih.gov/ij</a></td>
		</tr>
		<tr>
			<td>rainSTORM</td>
			<td>Laser Analytics Group (Cambridge) software</td>
			<td>&nbsp;</td>
			<td><a href="http://laser.cheng.cam.ac.uk/wiki/index.php/Resources" target="_blank">http://laser.cheng.cam.ac.uk/wiki/index.php/Resources</a></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Running rainSTORM</td>
			<td>&nbsp;</td>
			<td><a href="http://www.mathworks.co.uk/products/matlab/" target="_blank">http://www.mathworks.co.uk/products/matlab/</a></td>
		</tr>
		<tr>
			<td>*Example of self-built STORM/PALM microscopes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Also see reference 29 <a href="http://code.google.com/p/quickpalm/wiki/Tutorial_CustomMicroscope" target="_blank">http://code.google.com/p/quickpalm/wiki/Tutorial_CustomMicroscope</a></td>
		</tr>
		<tr>
			<td>*Commercial STORM/PALM super-resolution microscopes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Leica SR GSD, Nikon N-STORM, Vutara SR-200, Zeiss Elyra P.1, DeltaVision OMX Blaze (Monet Localization) Alternatives to self-built microscopes</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Materials &amp; Equipment</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Fisher</td>
			<td>VX70013065</td>
			<td>1; 2; 3; 4</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td>1.1; 2.1</td>
		</tr>
		<tr>
			<td>Dextran-Alexa fluor 647</td>
			<td>Invitrogen</td>
			<td>D-22914</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>General actin buffer</td>
			<td>Cytoskeleton Inc</td>
			<td>BSA01</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>Preformed-actin filaments</td>
			<td>Cytoskeleton Inc</td>
			<td>AKF99</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>Phallodin-Alexa fluor 647</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>TetraSpeck Microspheres (0.1 micron)</td>
			<td>Invitrogen</td>
			<td>T-7279</td>
			<td>3.1</td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Fisher</td>
			<td>VX31966021</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher</td>
			<td>VX10500064</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>Pen/Step</td>
			<td>Invitrogen</td>
			<td>15070063</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>Plastic dishes</td>
			<td>Invitrogen</td>
			<td>734-0006</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>EGF - Alexa fluor 647</td>
			<td>Invitrogen</td>
			<td>E35351</td>
			<td>4.3</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>4.3, 4.4</td>
		</tr>
		<tr>
			<td>Formaldehyde - 10% solution EM grade</td>
			<td>Polysciences</td>
			<td>04018-1</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C100</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma</td>
			<td>C4706</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>93352</td>
			<td>5.1</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>C7528</td>
			<td>5.2</td>
		</tr>
		<tr>
			<td>MEA-HCl</td>
			<td>Sigma</td>
			<td>M6500</td>
			<td>5.3</td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>Sigma</td>
			<td>G2289</td>
			<td>5.1; 5.2</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Reagents</td>
		</tr>
	</tbody>
</table>

test samples for optimizing storm super-resolution microscopy

暴风影音是比标准的荧光显微镜技术更好的分辨率高达10倍最近开发的超分辨率显微镜技术。然而，由于图像被获取以非常不同的方式比正常的，通过建立一个图像分子通过分子中，有在试图优化其图像采集的用户一些显著挑战。为了帮助这一进程，并获得更多的深入了解，暴风影音的工作，我们提出了3编制试验样品和采集和处理风暴30-50纳米之间的典型分辨率超分辨率图像的方法。通过将试验样品与使用免费提供的暴雨处理软件也能够获得大量的关于图像质量和分辨率的信息。使用这些度量它然后就可以从光学优化成像过程中，样品制备，染料的选择，缓冲液条件，以及图像获取设置。我们一那会导致图像质量差一些共同的问题，如横向漂移，其中图像获取和密度在样品移动有关，导致了“错误定位”现象的问题LSO显示的例子。

Watch this Scientific Journal Video about Test Samples for Optimizing STORM Super-Resolution Microscopy at JoVE.com

Test Samples for Optimizing STORM Super-Resolution Microscopy

我们描述了制备3个试样，以及如何可以被用来优化和评估STORM显微镜的性能。使用这些例子中，我们展示了如何获取原始数据，然后进行处理，以获得超分辨率图像约30​​-50 nm分辨率的细胞。

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

test-samples-for-optimizing-storm-super-resolution-microscopy

Research

JoVE Journal

Biology

31.0K 次观看.  National Physical Laboratory. 我们描述了制备3个试样，以及如何可以被用来优化和评估STORM显微镜的性能。使用这些例子中，我们展示了如何获取原始数据，然后进行处理，以获得超分辨率图像约30​​-50 nm分辨率的细胞。 

视频: Test Samples for Optimizing STORM Super-Resolution Microscopy

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

科研

生物学

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

三维超F-微丝高分辨率显微镜通过干涉光敏定位显微镜（iPALM）

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

高速超分辨速度显微镜在活原纤毛中的应用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to chromatic aberration and the imperfect alignment of cameras, images acquired in each channel may be shifted, and magnified, as well as rotated relative to each other in any of the three dimensions. With the classical calibration method, chromatic shifts are measured by multicolor beads attached to the surface of a coverslip, and a number of software are available to measure the chromatic shifts from such calibration samples. However, chromatic aberration can vary with depth, change with observation conditions and be induced by the biological sample itself, thus hindering determination of the true amount of chromatic shift in the sample of interest and across the volume. Correcting chromatic shifts at higher accuracy is particularly relevant for super-resolution microscopy where only slight chromatic shifts may affect quantitative analyses and alter the interpretation of multicolor images. We have developed an open-source software Chromagnon and accompanying methods to measure and correct 3D chromatic shifts in biological samples. Here we provide a detailed application protocol that includes special requirements for sample preparation, data acquisition, and software processing to measure chromatic shifts in biological samples of interest.

High-Accuracy Correction of 3D Chromatic Shifts in the Age of Super-Resolution Biological Imaging Using Chromagnon

Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.

使用色马格龙的超分辨率生物成像时代3D色变的高精度校正

Quantitative multicolor fluorescence microscopy relies on the careful spatial matching of color channels acquired at different wavelengths. Due to ...

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 ...