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?...

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>Microscope frame</td><td>Nikon</td><td>Ti-U</td><td></td></tr><tr><td>60X oil immersion TIRF objective with 1.49 NA</td><td>Nikon</td><td>CFI Apo TIRF 60x H</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>Opto Engine</td><td>MGL-FN-561</td><td></td></tr><tr><td>405 nm laser</td><td>Coherent</td><td>CUBE-405 50mW</td><td></td></tr><tr><td>561 nm dichroic mirror</td><td>Semrock</td><td>&amp;nbsp;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 CO&lt;sub&gt;2&lt;/sub&gt; controlled stage</td><td>Tokai Hit</td><td>INUBG2ATW-PLAM</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&#039;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 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?...

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

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

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫与感染

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

生物化学

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 ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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

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

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

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

活体哺乳动物细胞器中单膜蛋白的多色定位显微术

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

To prepare a sample for imaging, plate about 5.5 x 104 stable expression cells per well of an 8-well glass-bottomed chamber slide, in 350 microliters of phenol red-free DMEM. Use fresh paraformaldehyde with glutaraldehyde to fix the cells, and after replacing the fixative with PBS or imaging buffer, vortex 100-nanometer gold particles, and add 35 microliters per well for tracking stage drift during imaging.
Approach the microscope and power on the 405 and 561-nanometer lasers. Keeping the shutters closed at this point, ensure the 561-nanometer dichroic mirror and notch filter are in place. Open the image acquisition software.
Turn on the EMCCD camera and allow it to cool down, and set the exposure time to 100 milliseconds and the EMCCD gain to an appropriate value. Add immersion oil to the objective, and secure the sample on the microscope stage. Next, with either brightfield or the 561-nanometer laser, bring the sample into focus.
For imaging Ras and other membrane proteins, use a 60X apochromat TIRF objective with a 1.49 numerical aperture, and bring the microscope into TIRF configuration.
Adjust the excitation laser so that it is off-centered when hitting the back aperture of the TIRF objective, which will cause the laser to deflect upon reaching the coverslip buffer interface. Keep adjusting the laser incident position until the critical angle is reached and the laser is being reflected back.
Search for a cell to image with several gold particles in view. Then, set a region of interest that encloses the cell or a region of it and the gold particles.
In case sufficient activation is already occurring because of high expression levels, begin acquisition with the 405-nanometer laser off and the 561-laser on. Otherwise, turn on the 405-nanometer laser at the lowest factory power setting, and increase it gradually until there are tens of molecules per frame or so that single molecules are well-separated.
As data acquisition continues, gradually increase the 405-nanometer laser power to keep the spot density roughly constant. Continue image acquisition until high 405 power does not initiate any more activation events.