使用双光镊和微流体进行单分子研究
Summary
使用玻璃桶、气密注射器、管道与流通池的稳定连接以及通过在注射器和管道之间放置切换阀来消除气泡，极大地促进了通过微流体室研究的视觉单分子生物化学。该协议描述了双光学陷阱，能够可视化DNA交易和分子间相互作用。
Abstract
视觉生物化学是一种强大的技术，用于观察单个酶或酶复合物的随机特性，这些特性在批量阶段研究中发生的平均中被掩盖。为了实现可视化，双光学镊子，其中一个陷阱固定，另一个是移动的，聚焦到位于倒置荧光显微镜载物台上的多流微流体室的一个通道中。光学镊子捕获荧光标记的DNA的单个分子，流体流过腔室并经过捕获的珠子，将DNA拉伸至B形（在最小力下，即0 pN），核酸在黑色背景下被观察为白色字符串。DNA分子从一个流移动到下一个流，通过平移垂直于流动的阶段，以受控方式启动反应。为了取得成功，具有光学透明通道的微流体装置与固定在注射泵中的玻璃注射器配合使用。最佳结果是使用永久粘合到流通池的连接器，机械刚性和耐化学腐蚀的管道，以及连接到开关阀，以消除禁止层流的气泡。
Introduction
在单分子水平上实时可视化蛋白质-DNA相互作用的能力为基因组稳定性提供了重要的见解1，2。除了一次处理一个DNA分子外，查看附近单个分子之间的交易的能力还提供了额外的洞察力3，4，5。操纵额外的DNA分子既需要额外的光学陷阱，也需要高质量的多通道微流体流通池6。
有几种方法可用于生成多个光学陷阱。这些包括振镜扫描镜、声光调制器和衍射光学器件，它们产生全息光镊4，7，8，9。通常，扫描镜和声光调制器会产生分时陷阱。在这里描述的设置中，单个Nd：YAG激光器的光束在偏振上分裂，然后振镜激光扫描镜控制所谓的移动陷阱的位置（图1）4。为了便于定位反射镜和滤光片，将捕获光束引导到显微镜物镜的后孔径，使用了HeNe激光器。这使得整体对准更容易，因为HeNe光束是肉眼可见的，而红外光束则不是。氦氖光束也更安全，可以减轻镜子和其他组件的定位压力。最初，该激光器的光束路径与1064 nm光束分开，但被引入相同的光束路径，然后进入显微镜物镜。一旦实现物理对准，就可以将 1064 nm 光束定位在 HeNe 光束的顶部，这可以通过使用红外观察器和各种光束成像工具来可视化光束位置和质量来实现。然后，引入扩束镜，并将所得的扩增红外光束对准物镜的后孔径。最后，移除物镜，并使用λ/2波片测量和调整每个偏振光束中的功率以使其相等（图1C）。返回物镜后也会进行功率测量，通常有53%的功率损耗。然而，有足够的功率在焦平面中形成稳定的固定和移动光学陷阱（图1D）。
为了对DNA交易进行成像，微流体流通池起着关键作用，因为它们允许在单分子水平上进行具有高空间和时间分辨率的受控测量（图2）。术语微流体是指在一个或多个通道中操纵流体的能力，尺寸范围为5-500μm10，11。术语流是指通道内的实际流体，通道是指一个或多个流体流在其中移动的物理通道。单通道流通池设计具有一个通用的物理通道，用于观察反应，并且通常仅存在一个流体流。因此，这些设计被称为单流流通池。相比之下，多流流通池被定义为一种微流体装置，其中两个或多个入口通道汇聚成一个公共物理通道（图2A）。在公共通道内，源自各个通道的流体流彼此平行流动，并保持分离，由于扩散，它们之间的混合很小（图2B）。在大多数实验设置中，单个泵以相同的速度将流体推入每个通道。相反，当使用边界转向时，三个或更多独立控制的泵将流体推动通过通道。然而，每个泵以不同的速度运行，但公共通道中的净流量恒定为12。这允许通过改变泵速来快速更换主通道组件。
除了层流，另一个关键因素是层流内的抛物线速度分布。最高流速出现在流的中间，最慢的流速出现在表面旁边（图2C）13。必须考虑该谱图以完全拉伸附着在流中的磁珠上的DNA分子，以实现荧光DNA的精确可视化和准确的单分子分析。在这里，DNA被拉伸到B形式，并在0pN的力下保持在适当的位置。为此，光学陷阱位置的聚焦应位于距底部盖玻片表面10-20μm的位置（图2D）。必须注意使DNA分子不会拉伸到B型以上，因为这会抑制酶反应。在典型的缓冲条件下，1 μm = 3，000 bp 的 DNA14。此外，通过从盖玻片捕获10-20μm，DNA复合物远离表面，从而最大限度地减少表面相互作用。
许多方法已被用于创建微流体设备通道，这些可以在实验室中完成，或者可以从商业来源购买流通池6，15，16，17。用于构建流通池的最佳材料必须是机械刚性、低荧光的光学透明和不受有机溶剂的影响6。通常，硼硅酸盐浮法玻璃或熔融石英用于长时间提供稳定的流动环境，适用于光学捕获、可视化和力检测。这些材料还允许使用非水溶剂（例如分光光度级甲醇）来简化表面润湿和去除气泡，并使用变性剂（例如6M盐酸胍）或洗涤剂来清洁流通池。最后，用于将流体引入流通池的方法从复杂的真空泵系统到单注射泵14，18，19，20，21，22，23，24，25，26，27。在这里描述的方法中，使用最多可容纳10个注射器的注射泵（图3A）。这为使用单通道流通池或具有多个入口通道的流通池提供了灵活性。在这里，使用三通道流通池，并使用聚醚醚酮（PEEK）管与注射泵上固定的注射器配合使用（图3A-C）。流体的流动由四通切换阀控制，从而最大限度地减少气泡进入流通池（图3A，D）。此外，建议使用具有坚硬玻璃壁和聚四氟乙烯（PTFE）涂层柱塞的汉密尔顿气密注射器，因为它们提供异常平稳的柱塞运动，这对于获得平稳的流动至关重要14，27。
在所描述的实验系统中，使用了具有两到五个入口通道的流通池。入口通道的数量由正在进行的实验决定。对于RecBCD和Hop2-Mnd1的研究，两个流道就足够了14，28。对于解旋酶，酶与DNA的自由端结合并翻译成含有镁和ATP的流以启动易位和解开。对于Hop2-Mnd1，光学捕获的DNA被翻译成含有蛋白质和缓冲液的相邻流体流±二价金属离子。使用三通道流通池可以捕获流1中的DNA，将DNA翻译成流2以允许蛋白质结合发生，然后在存在ATP的情况下将DNA翻译成流3，例如启动反应。上述方法的变体是在通道2中使用荧光标记的蛋白质，这导致流体流完全白色并排除DNA的可视化。当该分子被翻译成流3时，当反应开始时，蛋白质和DNA现在都可见。
使用四通切换阀来控制流体流量是消除流通池中气泡的系统的关键组成部分。气泡不利于稳定的流体流动，因为它们以不可预测的方式收缩和膨胀，导致流速快速变化并引入湍流。当阀门位于注射器和入口管之间时，当更换注射器时，通过切换阀门位置来断开流路。当新注射器就位时，可以手动压下柱塞，以便喷射出>6μL（阀门的死体积），这几乎完全消除了气泡。
将连接器连接到流通池通常是流通池使用中的限速步骤。我们描述了两种类型的连接器的使用：称为压接的可拆卸连接器和永久连接器（纳米端口组件）。可拆卸连接器易于粘附在流通池上，除了推荐的PTFE外，还可以使用这些连接器测试不同类型的柔性管。这是一种快速且经济高效的方法来测试管道和连接器，而不会牺牲更昂贵的玻璃流通池。相比之下，纳米端口组件是永久连接的，可承受高达1，000 psi的压力，并且在我们手中，它们的使用仅限于不同直径的PEEK管。这不是缺点，因为最好使用PEEK管。连接有永久组件的单个玻璃流通池可以重复使用 1 年以上，小心使用。
Protocol
Representative Results
Discussion
流动系统的仔细组装对于实验4，6的成功结果至关重要。该协议最具挑战性的方面之一是将连接器连接到玻璃表面。为此，我们使用以下两种方法：压配管接头和纳米端口组件。压接连接器很容易粘附在玻璃上，然后使用镊子将PTFE管推入预成型孔中。当需要更稳定的附着时，优选纳米端口组件的粘合。通过仔细使用和清洁，带有永久组件的单个玻璃流?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Bianco实验室的研究得到了NIH对PRB的GM100156和GM144414的支持。
Materials
| 100x objective | Leica | 506318 or 506038 | Oil immersion lenses; Imaging and optical trapping only; Plan APO objectives optimized for fluorescence imaging |
| 10X Objective | Leica | 506263 | Used to locate laser beams spots during alignment; to find focus and X-Y position in flow cell |
| 1 mm fluorescent beads | Bangs Labs | FSDG004 | Used for tap performance, focal position determination |
| 1 mm polystyrene beads | Bangs Labs | CPO1004 | Used for trap performance evaluation and binding to biotinylated molecules |
| 63x objective | Leica | 506081 | Used to locate laser beams spots during alignment and to find focus and X-y position in flow cell; can be used for optical trapping as it has an identical back aperture diameter to the 100X; oil immersion lens |
| Alignment laser | Lumentum | 1100 series | 10mW HeNe laser that is visible to the naked eye that is used to position optics |
| Beam alignment camera | Amscope | MU303 | A simple, inexpensive and software controlled camera for imaging of the beam position |
| Camera control and Image capture software | Hamamatsu | HCImage | Coordinates activities of the Lambda DG4 with the camera to facilitate rapid wavelength switching |
| Camera; Orca flash 4 | Hamamatsu | c13440-20cu | CCD camera for imaging of single-molecule experiments |
| C-mount for the beam alignment camera | Spot imaging solutions | DE50CMT | Provides optimal positioning of the camera for imaging of laser beams during alignment |
| C-mount for the Orca Flash 4 camera | Has a retainer ring to hold an IR blocking filter in place. This eliminates reflected IR beam from the optical traps and facilitates clearer imaging of trapped objects. | ||
| Cy5 fluorescence filter cube | Semrock | cy5-404a-lsc-zero | Used in conjunction with Lambda DG4 to image Cy5 only |
| Fitc-Txred fluorescence filter cube | Semrock | fitc/txred-2x-b-000 | Used in conjunction with Lambda DG4 to image FITC and TXRed |
| Fluidics tubing | Grace Bio | 46004 | PTFE tubing as an alternate to PEEK; works well on some flow cells. Can be used with PDMS flow cells or glass flow cells when Grace Bio fit tubing connectors are used |
| GFP fluorescence filter cube | Semrock | gfp-3035b-lsc-zero | Used in conjunction with Lambda DG4 to image GFP only |
| Glass flow cells | Translume | Custom | Clear flow channels for imaging (Fig. 2E) |
| Glass glue | Loctite | 233841 | Securely and easily bonds Nanoport assemblies to glass flow cells |
| Glass/PDMS sandwich flow cells | CIDRA Precision services | Custom design | Flow cells built according to your specifications; imaging channels are clear (Fig. 2C) |
| Hamilton Cleaning solution | Hamilton | 18311 | Gentle but efficient cleaning solution for glass flow cells; does not bubble when used carefully |
| Illumination system | Sutter Instrument | Lamda DG4 | Discontinued so recommend Lambda 721 |
| Illumination system | Sutter Instrument | Lamda DG4 | Discontinued so recommend Lambda 721 |
| Image analysis software | Media cybernetics | Image Pro Premiere | Analysis of images and single molecule tracking |
| Image analysis software | Fiji/NIH Image/Image J | Shareware | Analysis of images and single molecule tracking |
| Image display card | Melles Griot | 06 DLA 001 | Alternate product from Thorlabs: VRC5 |
| Immersion oil | Zeiss | 444960 | Immersol 518 F fluorescence free |
| Laser beam alignment tools | Thor labs | FMP05/M; dgo5-1500-h1; BHM1 | Used to ensure beams are horizontal and at the correct height |
| Laser beam viewer | Canadian Photonics labs | IR 3150 | Used to image IR beam spots on mirrors and targets |
| Laser power meter | Thor labs | Measurement of laser output as well as trap strength | |
| Laser safety glasses (HeNe) | Thor labs | LG7 or 8 | Blocks >3 OD units of light of wavelengths >600 nm |
| Laser safety glasses (IR) | Thor labs | LG11 | Blocks >7 OD units of light of wavelengths ³1000 nm |
| Mcherry fluorescence filter cube | Semrock | mcherry-a-lsc-zero | Used in conjunction with Lambda DG4 to image mcherry only |
| Microscope | Leica | DMIRE2 | DIC port removed to accommodate Dichroic trapping/alignment mirror |
| Microscope control software | UCSF/shareware | uManager | Controls the microscope, permits focal alignment of objectives as well as stage control |
| Nanoport assembly | IDEX | N333 | Connectors that are bonded to flow cells |
| Optical table support | Thor Labs | PA52502 | Active isolation table support |
| Optics and lenses | Solar TII | Various | Interference mirrors, telescopes and lenses custom designed for the system |
| PDMS flow cells | ufluidix | Custom | Flow cells built according to your specifications; imaging channels are clear (Figs. 2B and D) |
| PEEK tubing | IDEX | 1532 | Provides excellent connection to flow cells and switching valves |
| Pinkel fluorescence filter cube | Semrock | lf488/543/635-3x-a-000 | Used in conjunction with Lambda DG4 to image multiple fluorophores rapidly |
| Press fit tubing connectors | GraceBio | 46003 | Clear silicone connector with adhesive that binds well to glass |
| Scanning mirrors | GSI Lumonics | VM500 | Used to provide control of the second optical trap. GSI Lumonics no longer exists. Similar mirrors can be purchased from Cambridge Scientific |
| Stage | Leica | ||
| Stage micrometer | Electron Microscopy Sciences | 68042-08 | Provides on screen ruler for positioning of the beam and system calibration |
| Switching valves | IDEX | V-101T | Control direction of fluid flow and eliminate introduction of bubbles into flow cells |
| Syringe and valve manifold | Machine shop | None | Custom built |
| Syringe pump | Harvard Apparatus | PHD 2000 | Controls fluid flow through flow cells |
| Syringe pump software | Harvard Apparatus | 70-6000 | Flow control provides seamless, programmable control of fluid flow |
| Syringes | Hamilton | 81320 | Gas-tight, PTFE Luer Lock, glass barrels with Teflon-coated plungers |
| Table top | Thor Labs | T36H | Optical table top or breadboard |
| Trapping laser | Newport/Spectra Physics | J-series; BL106C | Nd:YAG laser; 1064 nm; 5W laser |
References
- Bianco, P. R., Lu, Y. Single-molecule insight into stalled replication fork rescue in Escherichia coli. Nucleic Acids Research. 49 (8), 4220-4238 (2021).
- Kaur, G., Lewis, J. S., van Oijen, A. M. Shining a spotlight on DNA: Single-molecule methods to visualise DNA. Molecules. 24 (3), 491 (2019).
- Dame, R. T., Noom, M. C., Wuite, G. J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature. 444 (7117), 387-390 (2006).
- Bianco, P. R., Bradfield, J. J., Castanza, L. R., Donnelly, A. N. Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis. Journal of Molecular Biology. 374 (3), 618-640 (2007).
- Amitani, I., Liu, B., Dombrowski, C. C., Baskin, R. J., Kowalczykowski, S. C. Watching individual proteins acting on single molecules of DNA. Methods in Enzymology. 472, 261-291 (2010).
- Brewer, L. R., Bianco, P. R. Laminar flow cells for single-molecule studies of DNA-protein interactions. Nature Methods. 5 (6), 517-525 (2008).
- Visscher, K., Brakenhoff, G. J., Krol, J. J. Micromanipulation by multiple optical traps created by a single fast scanning trap integrated with the bilateral Confocal scanning laser microscope. Cytometry. 14, 105-114 (1993).
- Vermeulen, K. C., Mameren, J. v. Calibrating bead displacements in optical tweezers using acousto-optic deflectors. Review of Scientific Instruments. 77 (1), 013704 (2006).
- Dufresne, E. R. Computer-generated holographic optical tweezers arrays. Review of Scientific Instruments. 72 (3), 1810 (2001).
- Squires, T. M., Quake, S. R. Microfluidics: fluid physics at the nanoliter scale. Reviews of Modern Physics. 77, 977-1026 (2005).
- Weibel, D. B., Whitesides, G. M. Applications of microfluidics in chemical biology. Current Opinion in Chemical Biology. 10 (6), 584-591 (2006).
- Tan, X., Mizuuchi, M., Mizuuchi, K. DNA transposition target immunity and the determinants of the MuB distribution patterns on DNA. Proceedings of the National Academy of Sciences of the United States of America. 104 (35), 13925-13929 (2007).
- Lima, R., Wada, S., Takeda, M., Tsubota, K., Yamaguchi, T. In vitro confocal micro-PIV measurements of blood flow in a square microchannel: the effect of the haematocrit on instantaneous velocity profiles. Journal of Biomechanics. 40 (12), 2752-2757 (2007).
- Bianco, P. R., et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature. 409 (6818), 374-378 (2001).
- Forget, A. L., Dombrowski, C. C., Amitani, I., Kowalczykowski, S. C. Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. Nature Protocol. 8 (3), 525-538 (2013).
- Streets, A. M., Huang, Y. Microfluidics for biological measurements with single-molecule resolution. Current Opinion in Biotechnology. 25, 69-77 (2014).
- Madariaga-Marcos, J., Corti, R., Hormeno, S., Moreno-Herrero, F. Characterizing microfluidic approaches for a fast and efficient reagent exchange in single-molecule studies. Scientific Reports. 10 (1), 18069 (2020).
- Grayson, P., Han, L., Winther, T., Phillips, R. Real-time observations of single bacteriophage lambda DNA ejections in vitro. Proceedings of the National Academy of Sciences of the United States of America. 104 (37), 14652-14657 (2007).
- Luo, G., Wang, M., Konigsberg, W. H., Xie, X. S. Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America. 104 (31), 12610-12615 (2007).
- Sia, S. K., Whitesides, G. M. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis. 24 (21), 3563-3576 (2003).
- Wuite, G. J. L., Davenport, R. J., Rappaport, A., Bustamante, C. An integrated laser trap/flow control video microscope for the study of single biomolecules. Biophysical Journal. 79 (2), 1155-1167 (2000).
- Kim, S., Blainey, P. C., Schroeder, C. M., Xie, X. S. Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA. Nature Methods. 4 (5), 397-399 (2007).
- Tanaka, H., Ishijima, A., Honda, M., Saito, K., Yanagida, T. Orientation dependence of displacements by a single one-headed myosin relative to the actin filament. Biophysical Journal. 75 (4), 1886-1894 (1998).
- Merenda, F., Andrews, D. L., Galves, E. J., Nienhuis, G., et al. Refractive multiple optical tweezers for parallel biochemical analysis in micro-fluidics. Proceeding of SPIE. , 6483 (2007).
- Brewer, L. R., Corzett, M., Balhorn, R. Protamine-induced condensation and decondensation of the same DNA molecule. Science. 286 (5437), 120-123 (1999).
- Ladoux, B., Quivy, J. P., Doyle, P. S., Almouzni, G., Viovy, J. L. Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions. Science Progress. 84, 267-290 (2001).
- Bianco, P. R., Bradfield, J. J., Castanza, L. R., Donnelly, A. N. Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis. Journal of Molecular Biology. 374 (3), 618-640 (2007).
- Pezza, R. J., Camerini-Otero, R. D., Bianco, P. R. Hop2-Mnd1 condenses DNA to stimulate the synapsis phase of DNA strand exchange. Biophysical Journal. 99 (11), 3763-3772 (2010).
- Rye, H. S., et al. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Research. 20 (11), 2803-2812 (1992).
