The authors thank Drs. Steven Chu and Joe W. Gray for helpful discussions, Henry Marr for his initial work on the BiFC-PALM project, and Alexis Shoemaker for her technical assistance. This work is supported by startup funds to X.N. from OHSU. Research in the Nan laboratory was also supported by NIH 5U54CA143836-05, the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI company.

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The authors have nothing to disclose.

附设一直是常用的技术，用于检测和细胞可视化质子泵抑制剂，而PALM是最近单分子超分辨率显微镜技术，使完整的生物样品的纳米级成像。附设与Palm的结合纳米的空间分辨率和单分子敏感性细胞内获得的生产者价格指数的选择性成像。附设掌扩展这两种技术的效用，而这表现在这项工作中，显示了在揭示其天然细胞环境的生产者价格指数的分子细节巨大潜力。尤其是，纳米级分辨率允许特定质子泵抑制剂的空间和时间调节在分子尺度上，这已在生物医学研究是一项挑战的详细的调查。然而，如前面提到的，附设-PALM由互补和慢发色团成熟的不可逆转的限制。 分裂PAmCherry1用于证明的原则和u效用附设掌的。 PAmCherry1已被广泛用于在PALM实验以其优良的光物理特性。使用PAmCherry1作为附设探针的一个额外的好处是低背景和高特异性和效率的蛋白质重构在37℃下耦合到质子泵抑制剂时。由于这些原因，分割PAmCherry1附设探针有望成为适用于涉及质子泵抑制剂高分辨率成像许多不同的生物学研究的宝贵资源。除了​​PAmCherry1，一个photoconvertible荧光蛋白，mEos3.2，也被拆分附设掌26。由于其发射光谱重叠，mEos3.2和PAmCherry1不能一起使用，但绿霸fps的最近报道27日，开放的可能性，生产者价格指数的双色纳米级的超高分辨率成像。 如在传统的附设在执行附设-PALM的关键步骤是该融合构建体的设计和克隆。事实上八个不同的表达构建具有要克隆和八个附设对具有在每次尝试实施附设和附设-PALM可以是一个繁琐的，如果不禁止，流程进行测试。然而，如图中的Ras-Raf的例子中，在感兴趣的蛋白质经常的生化和结构特性的先验知识可被用于指导融合构建的优化设计，待测试附设对的数目减少。尽管如此，目前还没有一个通用的准则，以确保附设（因此附设-PALM）的实验会工作;其中很大一部分仍然是一门艺术。 而附设和附设-PALM通常用于研究两种蛋白质之间杂二聚体的形成，但也可行的研究是否可使用这些方法，在这种情况下，两个候选蛋白将是相同的同型二聚体的形成。然而，如这样，两种蛋白具有相同的片段可交互，但是不会导致附设信号。尽管每个结构作为的竞争性抑制剂的其他，这种相互作用是可逆的，而导致成功的互补的相互作用会积累由于不可逆性。在一般情况下，有可能是在信号的降低，如果有每个构建体的表达水平之间的急剧差异。因此，这两个构建体的表达水平可能需要比较荧光强度（例如，野生型和突变型的同型二聚体样品之间）的内源表达水平时被确定。这也许是为什么附设实验通常与瞬时转染和结构的表达进行。 最后但并非最不重要的，所有附设实验必须有适当的控制来证实。如果附设信号存在 ​​时，有机会，该信号是非特异性（即片段重构自身和PPI的不是因为）。一个阴性对照是突变之一或已知两种蛋白扰乱相互作用，这将导致一个显著损失附设信号。如果这样的点突变是未知的，其它方法也可以使用，例如引入一个竞争性结合伴侣的蛋白之一，看看是否有在信号的伴随减少。 越是困难的情况排查缺乏附设信号，或假阴性的。在这样的情况下，它是重要的，包括在克隆过程中的阳性对照，如在转染GFP的控制。 Western印迹可以运行以检查构建体的表达。如果这些因素都是正常的，可能需要改变，例如接头长度和/或序列，或不同的附设的配置，应考虑其他因素。 至于对PALM部，许多参数必须在处理图像时被考虑。步骤2.4.2.1描述用于排序的坐标，并生成最终的PALM图像参数。第一两个参数，结合框架和结合距离，正如我们前面所述23定义如果两个本地化的事件都在100纳米，并出现小于8帧（100毫秒/帧）或〜0.8秒的间隔。如果是这样，那么它们将被认为从同一分子出现，其坐标将通过平均进行组合。暗状态与寿命超过0.8秒，PAmCherry1似乎是罕见的大得多;作为荧光团从一个长寿命暗状态恢复的情况下，是从一个不同的分子在近端距离发射荧光的区分两个定位事件被认为是产生于同一分子，只有当他们出现在一定数目的帧和物理距离。根据经验，一个"组合框"设置为8-12帧（0.8-1.2秒）似乎是我们的实验条件下最优的。最小RMS定义嵌合幅度和残留噪声后的标准偏差之间的最小比率嵌合。这些值记录在期间坐标提取.cor文件。 综上所述，附设-PALM结合附设和PALM的优点，使质子泵抑制剂在比以前已经取得了更多的细节调查。与考虑以上，并与适当的对照实验的因素中，附设-PALM应该是在广泛的生物设置研究质子泵抑制剂的有用工具。

蛋白质-蛋白质相互作用（质子泵抑制剂）是基本的生物学1和经由跨许多时间和长度尺度时空机制紧密调节。研究细胞在细胞膜上的信号，例如，已揭示动态，纳米级的空间隔室，以促进特定质子泵抑制剂和细胞过程2。因此，探讨生物系统的生产者价格指数有足够的空间和时间分辨率，高特异性和高灵敏度的能力，关键是要实现生物的机械理解。 双分子荧光互补（附设）是用于可视化的生产者价格指数在细胞与亚细胞分辨率和活细胞相容性3存在的少数手段之一- 5。该技术是相对简单的，并且涉及一个荧光蛋白为两个非荧光片段的分裂;当遗传标记，以两个相互作用的蛋白质和使得接近，所述片段可重组，以形成一个完整的荧光蛋白，产生荧光信号。如果设计得当，附设探头不应该自发重组在没有生产者价格指数。这样，在一个附设测定荧光信号将仅出现在质子泵抑制剂的存在下，使质子泵抑制剂以高特异性的直接可视化。使用荧光作为读出的附加好处是高灵敏度，亚细胞的分辨率，并具有高通量和高含量筛选测定法，等等的兼容性。对于这些好处，已经开发了许多基于不同母体的荧光蛋白质附设探针。正如在基于常规的光学显微镜所有其它检测技术，但是，附设的空间分辨率由光的衍射限于〜250纳米。这使得它的挑战，研究生产者价格指数的调控在纳米尺度，对此，由脂质筏6先前提到并举例第二拉斯纳米团簇7，是一个重要的尺度来理解许多细胞过程，如信号。 附设已结合光敏定位显微镜（PALM）8,9克服这个限制的空间分辨率的成像生产者价格指数10。 PALM是绕过通过随机激活和单荧光分子subdiffractive本地化荧光成像衍射极限的近超分辨率显微镜技术。在每个激活周期，荧光分子发出几百到几千光子并产生了一个单分子图像在检测器上。当图像衍射极限（〜在宽度250纳米），其质心可以以更高的精确度来确定，通常在10-50纳米根据所检测的光子数的顺序。通过激活和定位于样品中的每个荧光分子，高分辨率图像可以被重建。 PerformeD于活细胞，单分子跟踪（SMT-）PALM进一步允许采集的数千蛋白扩散轨迹从单电池 11。重要的是，PALM使用专门的荧光探针，如光活化荧光蛋白（PA-FPS）来实现随机启动。由于两个附设和Palm使用荧光蛋白，他们通过分割PAmCherry1，常用的PA-FP对于Palm，变成氨基酸159和160之间的两个片段合并。 基于分裂PAmCherry1的附设系统显示来自两个片段自发重建低背景信号。当遗传标记，以一对相互作用的蛋白质，这两个片段（RN =残基1-159; RC =蛋氨酸+残基160-236）再生有效，即使在37℃和无孵育在较低温度下，以形成完整的PAmCherry1蛋白，其是不为其他附设对12如父mCherry 13的情况。 Furthermo重，再溶解后PAmCherry1蛋白保留了母体PAmCherry1的光物理性质，诸如高对比度，中等光子产率，和快速光活化，等等，这对于精确的单分子的定位和高分辨率的PALM成像是至关重要的。 在这个协议中，利用附设掌的成像U2OS细胞的Ras-Raf的交互，采用分体式PAmCherry1（ 图1A）中描述。第一步是设计构建用于表达PAmCherry1片段（ 即 ，RN和RC）和感兴趣的蛋白质之间的融合蛋白。从理论上讲，对于每对候选蛋白（A和B）的，有八个对融合蛋白进行测试:RN-A / RC-B; RN-A / B-RC; RC-A / RN-B; RC-A / B-RN; A-RN / RC-B; A-RN / B-RC; A-RC / RN-B;和A-RC / B-RN。这个过程通常可以通过考虑候选蛋白的结构或生物化学特性被简化。在的Ras的情况下，该蛋白质是翻译后修饰的C-末端CAAX盒（C =半胱氨酸; A =脂族; X =任何），在这之后AAX基序被裂解掉。因此，RN或RC只能融合到的Ras的N-末端;这降低了融合蛋白对四个（图1B）的数目。对于Raf的，所述的Ras结合结构域（RBD残基51-131）的使用，并且能够进行标记两端。生成这四个融合配置:RN-KRAS / RC-RAF RBD; RN-KRAS / RAF RBD-RC; RC-KRAS / RN-RAF RBD;和RC-KRAS / RAF RBD-RN。 此外，片段和所关心的蛋白之间的连接体将需要被考虑。约10个氨基酸柔性接头常常被用来作为它提供了足够的自由互补发生。一个这样的接头是（GGGGS）×2，虽然有许多人已成功地应用，包括从一个多克隆位点（MCS）14生成的随机序列。所述接头的长度可能需要被优化，取决于不感兴趣的蛋白质和当相互作用及其取向的大小克。 所述PAmCherry1片段被包含在一小克隆骨干具有侧翼的MCS（参见材料清单）。目的基因可以通过限制性位点或具有结扎无关的方法插入。之后克隆和序列验证，表达盒转移到使用重组酶反应中，克隆过程具有高保真和高效率的表达载体。 接着，将得到的表达构建体转染到靶细胞系，或者，如果一个稳定的细胞系是理想的，包装成慢病毒为感染靶细胞系。瞬时转染允许附设配置快速验证，但是潜在的问题必须注意。使用的化学品经常染强调细胞，导致高自发荧光;而采用全内反射荧光（TIRF）microsco的吡啶可以通过限制所述照明体积，TIRF理想当质子泵抑制剂发生在细胞膜上缓解这个背景信号。此外，瞬时转染往往导致高水平的蛋白表达的，远超过那些内源蛋白质，这可能会导致在检测所述质子泵抑制剂工件。因此，建议的稳定细胞株后的适当附设配置已通过初步测试被确定来建立。稳定细胞株也有通过多西环素诱导15为可调表达的潜力。 感染后，将细胞进行选择用的抗生素，通常嘌呤霉素和新霉素，每个构建体。对于样品制备，图像采集和数据分析的后续步骤将在详细的协议进行说明。 采用这种方法，纳米簇中的的Ras-Raf的RBD附设-PALM图像形成例行观察（图2A &lt;/ STRONG&gt;）。一致，对的Ras-Raf的RBD SMT-PALM轨迹显示在扩散状态（图2B-D）的一个非均匀分布。这些结果表明，在多个状态存在于细胞膜上的Ras-Raf的络合物，据推测为单体和簇，具有潜在的生物学意义。这项工作表明在质子泵抑制剂在细胞与纳米空间分辨率和单分子灵敏度，这将是很难获得与常规附设，荧光共定位，或荧光共振能量转移（FRET）的选择性成像附设-PALM的功率。 当设计附设实验和解释的结果，要记住的附设过程是不可逆在大多数情况下，包括分裂PAmCherry1是重要的。一旦两个片段结合并形成一个完整的PAmCherry1蛋白，这两个片段之间的联系，因此，PPI成为永久性的。这限制了使用附设和附设-PAL的M表示监视质子泵抑制剂的动力学（即，结合动力学，而不是蛋白质复合物一旦被形成在扩散动力学），有时甚至有可能导致到PPI络合物的错误定位。 一个附加的需要考虑的因素在设计附设-PALM实验是延迟在发色团成熟所固有的荧光蛋白质。一旦两种蛋白质相互作用和在FP片段聚集在一起，它们通常重折叠的秒（60秒的一半时间为EYFP 3）的数量级。然而，随后的发色团成熟和荧光信号开发分钟的量级。虽然荧光可在拆分 ​​金星，快速折叠YFP变种重建后10分钟内检测，半时间成熟，一般往往是约60分钟16。分裂PAmCherry1观察到有类似的速率。因此，附设及附设掌是监视实时PPI动力学目前糟糕的选择;其他的我thods，如FRET和最近开发的二聚化相关的荧光蛋白17，可能更适合用于此目的。

<table>
	<tbody>
		<tr>
			<td>TIRF Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60X oil immersion TIRF objective with 1.49 NA</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>&#160;Di01-R405/488/561/635-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm filter</td>
			<td>Semrock</td>
			<td>FF01-525/45-25</td>
			<td></td>
		</tr>
		<tr>
			<td>405/561 nm notch filter</td>
			<td>Semrock</td>
			<td>NF01-405/488/568-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature and CO2 controlled stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry1_1-159-MCS</td>
			<td>Addgene</td>
			<td>60545</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry160-236-MCS</td>
			<td>Addgene</td>
			<td>60546</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.2-DEST</td>
			<td>Life Technologies</td>
			<td>12489-019</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-DEST</td>
			<td>Addgene</td>
			<td></td>
			<td>http://www.addgene.org/Eric_Campeau/</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-531</td>
			<td></td>
		</tr>
		<tr>
			<td>In-Fusion HD Cloning</td>
			<td>Clontech</td>
			<td>639649</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Clonase</td>
			<td>Life Technologies</td>
			<td>11791</td>
			<td></td>
		</tr>
		<tr>
			<td>Vira Power Lentivirus Packaging</td>
			<td>Life Technologies</td>
			<td>K497500</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE Transfection Reagent</td>
			<td>Roche</td>
			<td>13873800</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Scientific</td>
			<td>155409</td>
			<td>#1.5 glass bottom dishes</td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with phenol red</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM no phenol red</td>
			<td>Life Technologies</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovit&#39;s L-15, no phenol red</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Life Technologies</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral concentrator</td>
			<td>Clontech</td>
			<td>631231</td>
			<td></td>
		</tr>
		<tr>
			<td>Retroviral concentrator</td>
			<td>Clontech</td>
			<td>631455</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>BD Biosciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A11138</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Calbiochem</td>
			<td>345812</td>
			<td>Neomycin</td>
		</tr>
		<tr>
			<td>Doxycyline</td>
			<td>Fisher</td>
			<td>BP2653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Sigma</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm gold particles</td>
			<td>BBI Solutions</td>
			<td>EM.GC100</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Life Technologies</td>
			<td>10977</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn1</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Midiprep kit</td>
			<td>Macherey-Nagel</td>
			<td>740410</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-137</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL&#160; polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>GeneMate</td>
			<td>C-3328-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Life Technologies</td>
			<td>15544</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD Biosciences</td>
			<td>244610</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Fisher</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells</td>
			<td>Life Technologies</td>
			<td>C4040</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

所示的附设-PALM例子是KRAS G12D突变体与RAS相互作用结合CRAF的结构域（RBD）（图1A）。如所讨论的，在RN或RC片段并未标记到的Ras的C-末端，因为它会破坏膜定位的Ras，因此生物活性。这减少了可能的组合，从8至4（图1B）。这四个组合被引入到使用慢病毒感染U2OS细胞。将细胞固定在协议中详述的，并使用PALM设置附设信号进行了评价。细胞与阳性附设信号通过在荧光的突然增加来识别时的405nm的激光被接通;在561 nm激光是在整个成像实验。在荧光这种突然变化是由于附设-重构PAmCherry1的基于照明的光活化的405nm的激光。使用这种方法在样品中多个区域进行了评价和附设信号强度进行了计算前平均增加像素强度和高功率脉冲从405nm的激光后。 正如图1C，出了四种可能的结构中，两种配置（即RN-的Ras / RC-Raf的RBD和RN-的Ras / Raf的RBD-RC）具有较强的附设信号。另一种配置（RC-RAS / RAF RBD-RN）的微弱信号，和一个没有信号。对于所有后续的实验中，在RN-的Ras / Raf的RBD-RC配置（右上图 1C）中的使用。作为阴性对照，点突变（R89L）中的Raf的的Ras结合结构域引入并使用相同的配置的附设效率进行了测试。这种突变被称为破坏的Raf结合的Ras;一致，突变大大降低了附设信号（图1D）。 因为通过附设产生PAmCherry1分子显示出类似的光活化性为原始PAmCherry1 10，这允许附设的样品使用相同的实验设置与母PAmCherry1 PALM成像。细胞与感染RN-Ras和Raf的RBD-RC后强附设信号的一例的PALM图像示于图2A中。 放大时（图2A底左和插图，与盒装放大的区域），从个人分子及其纳米级的空间分布可以清楚地看到。值得注意的是，在这些PALM图像中的每个点代表一个推定的Ras-Raf的RBD络合物具有标记一个PAmCherry1分子。为了比较，右侧图2B的 ，模拟的视图相同的字段，其中该集群成为遮去的低分辨率图像。中的Ras-RAF RBD集群的观察结果与以前的意见，位于Ras和Raf的集群行为是一致的。 随着活细胞表达附设重组PAmCherry1，SMT-PALM是PERFORmed指获得个体的Ras-Raf的RBD络合物扩散轨迹，类似于先前描述的10。根据与561 nm和405 nm激光连续照明，个人PAmCherry1分子随机切换，并进入黑暗的状态（这部分是由于漂白）前的最后几帧。在这一段时间内，所述分子显示出至少两个不同的扩散行为:一个动状态（图2B，分子1）和移动状态（图2B，分子2）。这两种类型的轨迹是清晰地呈现在（X，Y）在图2C中，其中多个分子的轨迹被记录示出曲线图。扩散状态的相同不均匀分布可以从连续帧之间的分子位移的直方图来推断。通常情况下，10,000 - 100000扩散轨迹可以从每个细胞被收购;这众多允许的T更严格的统计分析他扩散状态，例如与vbSPT算法25。 <img alt="图1" src="/files/ftp_upload/53154/53154fig1highres.jpg" width="700" /> 图1.附设-PALM实验设计的Ras-Raf的相互作用。（A）中的Ras是一种膜结合蛋白，其募集Raf的活性时。当非荧光分割PAmCherry1片段附着到Ras和Raf的，相互作用带来的片段一起和互补发生时，产生了功能性的荧光蛋白。 （ 乙 ）由于加标签的Ras在其C-末端会破坏它的膜定位，配置测试的Ras-Raf的附设-PALM数量从8减少到四个。 U2OS细胞表达了全内反射荧光显微镜拍摄的测试配置（C）图像。为了衡量每个配置的附设效率，细胞被赋予了405纳米的光，而同样的高剂量被激发与561纳米的光。 （d）将 R89L突变为英国皇家空军RBD大大降低了RN-KRAS G12D / CRAF RBD-RC配置的附设信号（右上C，N = 5-8）。误差棒是在SEM（D）。在（C）的比例尺为10μm。 RN = PAmCherry1 N末端 ​​片段，RC = PAmCherry1 C端片段，和RBD =的Ras结合结构域。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53154/53154fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53154/53154fig2highres.jpg" width="700" /> 图2.附设掌U2OS细胞表达RN-KRAS G12D / CRAF RBD-RC配置。（A）一个完整的手掌图像如上图所示。较低的面板被放大视图上的图像盒装区域所示。插图更高在各自的放大视图盒装地区的放大倍率。右侧的下面板（标TIRF）都在其左图像的表示，好像采取与衍射限制显微镜。 （B）中从一个活细胞SMT-PALM实验描绘缓慢（1）和快速（2）运动的Ras-Raf的复合物的连续帧。 （C）的输出从一个SMT-PALM实验示出的小区的小范围内的的Ras-Raf的络合物扩散轨迹。 （ 四）每RAS-Raf的复合框架位移从SMT-PALM实验的直方图。在（A）中比例尺为5μm顶部，500纳米底部，50纳米的插图， 和 （C），1微米。 RN = PAmCherry1 N末端 ​​片段，RC = PAmCherry1 C端片段，和RBD =的Ras结合结构域。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53154/53154fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table><tbody><tr><td></td><td>引物序列（5&#39;至3&#39;） </td></tr><tr><td>克隆的质粒，用于插入在N末端的反向</td><td> CATGGTACCGAGCTCCTGCAGC </td></tr><tr><td> RN N端前进</td><td> GTGAGCAAGGGCGAGGAGGATAA </td></tr><tr><td> RC N端前进</td><td> GGCGCCCTGAAGGGCGA </td></tr><tr><td>克隆的质粒，用于插入在C末端向前</td><td> TAAAAGGGTGGGCGCGCC </td></tr><tr><td> RN从C端反向</td><td> GTCCTCGGGGTACATCCGCTC </td></tr><tr><td> RC从C端反向</td><td> CTTGTACAGCTCGTCCATGCCG </td></tr></tbody></table> 表1 引物用于线性化含有PAmCherry1片段克隆质粒。含有RN和RC的克隆质粒可线性化，反向PCR在插入在N-或片段的C-末端的点。序列列5&#39;到3R17 ;. <table><tbody><tr><td></td><td>突出端对于感兴趣基因的引物（5&#39;至3&#39;） </td></tr><tr><td> N端插入RN或RC向前</td><td> GAGCTCGGTACCATG </td></tr><tr><td> N-末端插入RN反向</td><td> ACTTCCACCTCCACCACTACCTCCACCTCCCTCGCCCTTGCTCAC </td></tr><tr><td> N端插入RC反</td><td> ACTTCCACCTCCACCACTACCTCCACCTCCGCCCTTCAGGGCGCC </td></tr><tr><td> C端插入RN前锋</td><td> ATGTACCCCGAGGACGGAGGTGGAGGTAGTGGTGGAGGTGGAAGT </td></tr><tr><td> C端插入RC向前</td><td> GACGAGCTGTACAAGGGAGGTGGAGGTAGTGGTGGAGGTGGAAGT </td></tr><tr><td> C端插入RN或RC反</td><td> GCGCCCACCCTTTTA </td></tr></tbody></table> 表符合表1的引物2出挑感兴趣引物基因，产生的15个碱基对的同源性为结扎独立反应从底漆出挑。此外，突出端包含序列为（GGGGS）×2柔性接头。柔性接头序列可在表1中，而不是加入到引物。序列列5&#39;到3&#39;。

Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

photoactivated-localization-microscopy-with-bimolecular-fluorescence

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

9.8K 次观看.  Oregon Health and Science University. Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes. 

视频: Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

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

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

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

受体动力学通过单分子荧光显微镜的高分辨率时空分析

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

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...