使用一个荧光显微镜进行多种成像模式
Summary
在这里, 我们提出了一个实用的指南, 建立一个综合显微镜系统, 合并传统的 epi 荧光成像, 单分子检测的超分辨率成像和多色单分子检测, 包括单分子荧光共振能量转移成像, 成一种以经济高效的方式进行设置。
Abstract
荧光显微术是检测生物分子原位和实时监测其动力学和相互作用的有力工具。除了常规的免疫荧光显微镜外, 还开发了各种成像技术来实现特定的实验目标。一些广泛使用的技术包括单分子荧光共振能量转移 (smFRET), 它可以报告构象变化和分子相互作用与埃分辨率, 和单分子检测基于超分辨率 (SR) 成像, 与衍射有限显微镜相比, 它可以提高约十到多倍的空间分辨率。在这里, 我们提出了一个客户设计的集成系统, 它在一个显微镜中合并多种成像方法, 包括常规的 epi 荧光成像、基于单分子检测的 SR 成像和多色单分子检测,包括 smFRET 成像。通过切换光学元件, 可以轻松、可重复地实现不同的成像方法。这种设置很容易通过任何生物科学的研究实验室, 需要进行例行和多样化的成像实验, 以降低成本和空间相对于建立单独的显微镜为个人目的。
Introduction
荧光显微镜是现代生物科学研究的重要工具, 在许多生物实验室中都经常进行荧光成像。通过用荧光基团标记感兴趣的生物分子, 我们可以在显微镜下直接可视化它们, 并记录在体内或体外的定位、构象、相互作用和装配状态的时间依赖性变化。传统的荧光显微镜具有衍射有限的空间分辨率, 在横向方向上约为 200-300 nm, 在轴向1、2中为 500-700 nm, 因此仅限于100s 的成像。纳米到微米级。为了揭示分子组装或组织中更精细的细节, 开发了各种能打破衍射极限的 SR microscopies。用于实现 SR 的策略包括非线性光学效应, 如受激发射损耗 (STED) 显微镜3、4和结构化照明显微镜 (SIM)5、6、 7、随机检测单个分子, 如随机光学重建显微术 (风暴)8和光活化定位显微镜 (PALM)9, 以及两者的组合, 如 MINFLUX10。在这些 sr microscopies 中, 基于单分子检测的 sr 显微镜可以相对容易地从单分子显微镜设置中进行修改。通过重复激活和成像光敏荧光蛋白 (FPs) 或在感兴趣的生物分子标记的光交换染料, 空间分辨率可以达到 10-20 nm11。要获得实时的分子相互作用和构象动力学信息, 需要埃到纳米分辨率。smFRET12,13是实现这一决议的一种方法。通常, 根据感兴趣的生物学问题, 需要不同空间分辨率的成像方法。
通常, 对于每种类型的成像, 都需要特定的激励和/或发射光学配置。例如, 单分子检测最常用的照明方法之一是通过全内部反射 (TIR), 在这种情况下, 需要通过棱镜或通过物镜实现特定的激励角度。对于 smFRET 检测, 供体和受体染料的排放需要在空间上分离, 并定向到电子乘法、电荷耦合器件 (EMCCD) 的不同部分, 这可以通过一组反射镜和双色光束分配器实现。放置在排放路径中。对于三维 (3-D) SR 成像, 需要在发射路径中产生散光效应的光学元件, 如圆柱透镜14。因此, 自制或商业上可用的集成显微镜通常在功能上专门用于每种类型的成像方法, 并且在相同的设置下不灵活地在不同的成像方法之间切换。在这里, 我们提出了一个经济高效的混合系统, 提供三种不同成像方法之间的可调节和重复的开关: 传统的 epi-荧光成像, 衍射有限分辨率, 单分子检测 SR。成像和多色单分子检测, 包括 smFRET 成像 (图 1A)。具体而言, 此处提供的设置包含多色激励的光纤耦合输入激光器和激励路径中的商业照明臂, 允许对激励角度进行编程控制, 以便在 epi 模式和 TIR 模式之间切换。在发射路径中, 可拆卸的自制圆柱透镜盒放置在显微镜机身内, 用于3维 SR 成像, 在 EMCCD 摄像机前放置一个商用光束分配器, 可选择性地启用检测多个发射通道同时。
Protocol
1. 显微镜设计和组装 激励路径注: 激励路径包括激光器、差分干涉对比度 (DIC) 分量、显微镜本体及其照明臂。 准备一个隔振的光学表。例如, 结构阻尼表为 48 x 96 x 12 “为所有组件提供足够的空间。注: 在具有温度控制的房间内建立设置 (例如, 21.4 ±0.55 摄氏度)。温度稳定性对于保持光学对准至关重要。 安装一个显微镜机身, 配有用于光纤连接的照明?…
Representative Results
这种显微镜允许在不同的成像方法之间进行灵活和可重复的切换。这里我们展示了每个成像模块收集的样本图像。 图 5D显示了 SR 采集过程中闪烁的分子的 PSF。数以千计的这样的图像被重建, 以生成最终的 SR 图像 (图 5E)。图 5E显示了与图 7A</stro…
Discussion
这种混合显微镜系统无需购买多台显微镜。所有零件的总成本, 包括光学表、表安装人工、软件和工作站, 约为23万美元。定制加工部件, 包括 mag 透镜和3维透镜, 成本约为700美元 (成本取决于不同机构的实际费用)。用于基于单分子检测的 SR 显微显微镜的典型商业可用集成系统的成本超过30万美元 ~ 40万, 且不容易 smFRET 测量, 或在不使用白光的情况下为合理的视野提供 epi 成像。源。因此, 此处介绍的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
樱承认塞尔学者计划和 NIH 主任的新创新奖的支持。作者承认保罗 Selvin 的实验室 (伊利诺伊大学香槟分校) 为定位3维透镜提供了有用的建议。
Materials
| Nikon Ti-E microscope stand | Nikon | Ti-E | |
| Objective lens | Nikon | 100X NA 1.49 CFI HP TIRF | |
| Microscopy imaging software | Nikon | NIS-Elements Advanced Research/HC | HC includes "JOBS" module, the programmed acquisition module being used for SR imaging. |
| The illumination arm | Nikon | Ti-TIRF-EM Motorized Illuminator Unit M | This arm has a slot for a magnification lens |
| Analyze block | Nikon | Ti-A | This is installed in the filter turret. |
| Z-drift correction system | Nikon | PFS | This system is composed by the stepmotor on the objective nosepiece, IR LED, and a detector. |
| Optical table top | TMC | 783-655-02R | |
| Optical table bases | TMC | 14-426-35 | |
| 647 nm laser | Cobolt | 90346 (0647-06-01-0120-100) | Modulated Laser Diode 647nm 120mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit) |
| 561 nm laser | Coherent | 1280721 | OBIS 561nm LS 150mW Laser System |
| 488 nm laser | Cobolt | 90308 (0488-06-01-0060-100) | Modulated Laser Diode 488nm 60mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit) |
| 405 nm laser | Crystalaser | DL405-025-O | 405 (+/-5)nm, 25mW, Circular , M2 <1.3, Low Noise, CW, TTL up to 20MHz. 2 BNC connectors for TTL & Analog adjust |
| Heat sink | Cobolt | 11658 (HS-03) | Two units, Heat sink without fan HS-03, Heat sink for 647 nm and 488 nm lasers |
| Heat sink | Coherent | 1193289 | Obis heat sink with fan, 165 x 50 x 50 mm for the 561 nm laser |
| CAB-USB-miniUSB | Cobolt | 10908 | Two units, communication cable for 647 nm and 488 nm lasers |
| aluminum for height adjustment | McMaster-Carr | 9146T35 | Multipurpose 6061 Aluminum, Rectangular Bar, 4MM X 40MM, 1' Long for raising 561 nm laser |
| aluminum for height adjustment | McMaster-Carr | 8975K248 | Multipurpose 6061 Aluminum, 1-1/4" Thick X 3" Width X 1' Length for raising 405 nm laser |
| BNC cable | L-com | CC58C-6 | RG58C Coaxial Cable, BNC Male / Male, 6.0 ft |
| BNC adapter | L-com | BA1087 | Coaxial Adapter, BNC Bulkhead, Grounded |
| SMA to BNC Adapter | HOD | SMA-870 | Cobolt MLD lasers have SMA interface, so this adapter is used for BNC connection. |
| SMB to BNC Adapter | Fairview Microwave | FMC1638316-12 | SMB Plug to BNC Female Bulkhead Cable RG316 Coax in 12 Inch for Coherent Obis lasers |
| Data Acquisition Card | National Instruments | PCI-6723 | 13-Bit, 32 Channels, 800 kS/s Analog Output Device for controlling lasers, DIC LED, and etc |
| Barrier Filter Wheel controller | Sutter Instrument | Lambda 10-B | Optical Filter Changer |
| Emission Splitter | Cairn | OptoSplit III | |
| Dichroic beamsplitter | Chroma | T640LPXR-UF2 | Dichroic beamsplitter separating red emission from green emission in OptoSplit III |
| Dichroic beamsplitter | Chroma | T565LPXR-UF2 | Dichroic beamsplitter separating green & red emission from blue emission in OptoSplit III |
| Emission filter | Chroma | ET700/75M | Two units, Emission filter for red emission (like Alexa Fluor 647) in OptoSplit III as well as in the Barrier filter wheel |
| Emission filter | Chroma | ET595/50M | Two units, Emission filter for yellow/green emission (like Cy3B) in OptoSplit III as well as in the Barrier filter wheel |
| Emission filter | Chroma | ET525/50M | Two units, Emission filter for blue emission(like Alexa Fluor 488/GFP) in OptoSplit III as well as in the Barrier filter wheel |
| Emission filter | Semrock | FF02-447/60-25 | Emission filter for violet emission (like DAPI/Alexa Fluor 405), installed in the Barrier filter wheel |
| Dichroic beamsplitter | Chroma | zt405/488/561/647/752rpc-UF3 | Multiband dichroic beam splitter for 647, 561, 488, and 405 nm laser excitations inside of the microscope body |
| DAPI Filter set | Chroma | 49000 | installed in the microscope body |
| Nikon laser/TIRF filtercube | Chroma | 91032 | |
| 590 long pass filter | Chroma | T590LPXR-UF1 | for combining 647 nm laser and 561 nm laser |
| 525 long pass filter | Chroma | T525LPXR-UF1 | for combining already combined 647 nm and 561nm lasers with 488 nm laser |
| 470 long pass filter | Chroma | T470LPXR-UF1 | for combining already combined 647 nm, 561 nm and 488 nm lasers with 405 nm laser |
| Laser clean-up filter (647) | Chroma | zet640/20x | for cleaning up other wavelengths from the 647 nm laser |
| Laser clean up filter (488) | Semrock | LL01-488-25 | for cleaning up other wavelengths from the 488 nm laser |
| LED light source | Excelitas | X-Cite120LED | used only for DAPI imaging |
| Mirror mount | Newport | SU100-F3K | |
| Optical posts | Newport | PS-2 | |
| Clamping fork | Newport | PS-F | |
| Power Meter | Newport | PMKIT | For measuring laser power |
| Dichroic beamcombiner mount | Edmund Optics | 58-872 | C-Mount Kinematic Mount, for holding dichroic beamcombiners in the laser excitation assembly |
| Retaining ring | Thorlabs | CMRR | used for dichroic beamcombiner mounts |
| Fiber Adapter Plate | Thorlabs | SM1FC | FC/PC Fiber Adapter Plate with External SM1 (1.035"-40) Thread |
| Z-axis translational mount | Thorlabs | SM1Z | Z-Axis Translation Mount, 30 mm Cage Compatible |
| Achromatic Doublet lens | Thorlabs | AC050-008-A-ML | Ø5 mm, Mounted Achromatic Doublets, AR Coated: 400 – 700 nm |
| Cage Plate | Thorlabs | CP1TM09 | 30 mm Cage Plate with M9 x 0.5 Internal Threads, 8-32 Tap |
| Cage Assembly Rod | Thorlabs | ER4 | Cage Assembly Rod, 4" Long, Ø6 mm |
| Cage Mounting Bracket | Thorlabs | CP02B | 30 mm Cage Mounting Bracket |
| Single mode optical fiber | Thorlabs | P5-405BPM-FC-2 | Patch Cable, PM, FC/PC to FC/APC, 405 nm, Panda, 2 m |
| Multi mode optical fiber | Thorlabs | M42L01 | Ø50 µm, 0.22 NA, FC/PC-FC/PC Fiber Patch Cable, 1 m |
| Achromatic Doublet lens (mag lens) | Thorlabs | ACN127-025-A | ACN127-025-A – f=-25.0 mm, Ø1/2" Achromatic Doublet, ARC: 400-700 nm , a concave lens in the "mag lens" |
| Achromatic Doublet lens (mag lens) | Thorlabs | AC127-050-A | f=50.0 mm, Ø1/2" Achromatic Doublet, ARC: 400-700 nm, a convex lens in the "mag lens" |
| Retaining ring | Thorlabs | SM05PRR | SM05 Plastic Retaining Ring for Ø1/2" Lens Tubes and Mounts, for "mag lens" |
| Nylon-tipped screw | Thorlabs | SS3MN6 | M3 x 0.5 Nylon-Tipped Setscrew, 6 mm Long, for holding "3D lens" |
| 3D lens | CVI Laser Optics | RCX-25.4-50.8-5000.0-C-415-700 | f=10 m, rectangular cylindrical lens |
| EMCCD camera | Andor | iXon Ultra 888 | |
| 100 nm multichannel beads | Thermo | T7279, TetraSpeck microspheres | |
| red dye | Thermo | Alexa Fluor 647 | |
| yellow-green dye | GE Healthcare | Cy3 | |
| green dye | GE Healthcare | Cy3B | |
| blue dye | Thermo | Alexa Fluor 488 |
References
- Lipson, S. G., Lipson, H., Tannhauser, D. S. . Optical physics. , (1995).
- Török, P., Wilson, T. Rigorous theory for axial resolution in confocal microscopes. Optics Communications. 137 (1-3), 127-135 (1997).
- Klar, T. A., Hell, S. W. Subdiffraction resolution in far-field fluorescence microscopy. Optics Letters. 24 (14), 954-956 (1999).
- Hell, S. W., Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optics Letters. 19 (11), 780-782 (1994).
- Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy. 198 (2), 82-87 (2000).
- Gustafsson, M. G. L., et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophysical Journal. 94 (12), 4957-4970 (2008).
- Schermelleh, L., et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science. 320 (5881), 1332-1336 (2008).
- Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods. 3 (10), 793-795 (2006).
- Betzig, E., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313 (5793), 1642-1645 (2006).
- Balzarotti, F., et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science. 355 (6325), 606-612 (2017).
- Vaughan, J. C., Jia, S., Zhuang, X. Ultrabright photoactivatable fluorophores created by reductive caging. Nature Methods. 9 (12), 1181-1184 (2012).
- Ha, T., et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proceedings of the National Academy of Sciences of the United States of America. 93 (13), 6264-6268 (1996).
- Roy, R., Hohng, S., Ha, T. A practical guide to single-molecule FRET. Nature Methods. 5 (6), 507-516 (2008).
- Huang, B., Wang, W., Bates, M., Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science. 319 (5864), 810-813 (2008).
- Hua, B., et al. An improved surface passivation method for single-molecule studies. Nature Methods. 11 (12), 1233-1236 (2014).
- Fei, J., et al. RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA. Science. 347 (6228), 1371-1374 (2015).
- Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods. 5 (10), 877-879 (2008).
- Stracy, M., Kapanidis, A. N. Single-molecule and super-resolution imaging of transcription in living bacteria. Methods. 120, 103-114 (2017).
- Wang, S., Moffitt, J. R., Dempsey, G. T., Xie, X. S., Zhuang, X. Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proceedings of the National Academy of Sciences of the United States of America. 111 (23), 8452-8457 (2014).
- Sekar, R. B., Periasamy, A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. The Journal of Cell Biology. 160 (5), 629-633 (2003).
- Shi, X., et al. Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nature Cell Biology. 19 (10), 1178-1188 (2017).
- Youn, Y., Ishitsuka, Y., Jin, C., Selvin, P. R. Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy. Optics Express. 26 (2), 1670-1680 (2018).
Cite This Article
Park, S., Zhang, J., Reyer, M. A., Zareba, J., Troy, A. A., Fei, J. Conducting Multiple Imaging Modes with One Fluorescence Microscope. J. Vis. Exp. (140), e58320, doi:10.3791/58320 (2018).