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
Summary
传统的BODIPY结合物可用于活细胞单分子定位显微镜（SMLM），其瞬时成型、红移地面状态变暗器。我们提出了优化的SMLM协议，以跟踪和解决活哺乳动物和酵母细胞中的亚细胞中性脂质和脂肪酸在纳米长度尺度上。
Abstract
单分子定位显微镜（SMLM）技术克服了传统荧光显微镜的光衍射极限，能够以±20nm的精度解决细胞内结构和生物分子的动态。SMLM 的先决条件是荧光器，它从黑暗状态过渡到荧光状态，以避免其点扩散函数在数千个数据采集帧中每个帧中的分时重叠。BODIPYs 是成熟的染料，在传统显微镜中使用大量结合物。红移BODIPY地面状态变暗器（DII）的瞬态形成导致明亮的单分子发射，使单分子局部显微镜（SMLM）成为可能。在这里，我们提出了一个简单但通用的SMLM协议与传统的BODIPY结合在活酵母和哺乳动物细胞。此过程可用于获取超分辨率图像并跟踪单个 BODIPY-DII状态，以提取 BODIPY 偶联的时空信息。我们应用这个程序来解决脂质滴（LDs）、脂肪酸和在纳米长度尺度上活的酵母和哺乳动物细胞中的脂质脂滴（LD）、脂肪酸和淋索体。此外，我们演示了 BODIPY 染料与其他荧光探头结合使用时的多色成像功能。我们的代表性结果表明，在喂养和禁食条件下，酵母中BODIPY脂肪酸和中性脂质的空间分布和流动性存在差异。这种针对 SMLM 的优化协议可用于数百种商用 BODIPY 偶联，是研究纳米尺度生物过程的有用资源，远远超出本工作的应用范围。
Introduction
单分子定位显微镜（SMLM）技术，如随机光学重建显微镜（STORM）和光活化定位显微镜（PALM），已成为生成超分辨率图像的方法，其信息超过Abbe的光衍射极限11，2，2并跟踪单生物分子33，44的动态。与 SMLM 兼容的探头要求之一是能够随时控制活动荧光波的数量，以避免其点差函数 （PSF） 的空间重叠。在数千个数据采集帧中，通过安装其相应的点扩散功能，确定每个荧光荧光波的定位，精度为±20 nm。传统上，氟脑的开离闪烁是通过随机光切换11，2，52,5或化学诱导的内在闪烁6加以控制。其他方法包括荧光原在瞬态结合时诱导激活氟原蛋白77、8，8以及标记的DNA寡聚物在总内部反射荧光（TIRF）或光片激发9中的可编程结合。最近，我们报道了SMLM10的新颖和通用的标签策略，其中先前报道的红移二恶热（DII）状态的传统硼二丙甲甲（BODIPY）结合11，12，13是瞬时形成，并成为特别兴奋和检测与红移波长。11,12,13
BODIPYs是广泛使用的染料与数百种变种，专门标记亚细胞隔间和生物分子14，15，16。14,15,16由于其易用性和适用于活细胞，BODIPY 变体在商业上可用于传统荧光显微镜。在这里，我们描述了一个详细和优化的协议，如何将数以百计的商用 BODIPY 联结物用于活细胞 SMLM。通过调整BODIPY单体浓度，优化激发激光功率、成像和数据分析参数，在活细胞中获得高质量的超分辨率图像和单分子跟踪数据。当在低浓度（25-100 nM）下使用时，BODIPY偶联剂可同时用于红移通道中的SMLM和传统发射通道中的相关传统荧光显微镜。可以分析获得的单分子数据，以量化不动结构的空间组织，提取活细胞17中分子的扩散状态。具有绿色和红色两种形式的 BODIPY 探头，可在与其他兼容荧光波的正确组合中使用多色成像。
在本报告中，我们提供了一个优化的协议，用于使用BODIPY-C 12、BODIPY（493/503）、BODIPY-C12红色和多种颜色的色解解解器绿色来获取和分析活细胞SMLM数据。12我们以±30nm的分辨率解决活酵母和哺乳动物细胞中的脂肪酸和中性脂质。我们进一步证明，酵母细胞根据它们的代谢状态调节外部添加脂肪酸的空间分布。我们发现，在喂食条件下，在内质视网膜（ER）和脂液（LD）中加入的BODIPY-脂肪酸（FA）局部化，而BODIPY-FA在禁食后在血浆膜中形成非LD簇。进一步扩展了该技术在活体哺乳动物细胞中图像的脂体和LD的应用。我们使用传统的 BODIPY 联偶联体优化的 SMLM 协议是利用无数可用的 BODIPY 偶联物在纳米尺度上研究生物过程的有用资源。
Protocol
Representative Results
Discussion
在此协议中，我们演示了如何使用传统的 BODIPY 偶联来获取具有空间分辨率级级改进的 SMLM 图像。该方法基于利用先前报告的红色移位DII状态的传统BODIPY染料，这些状态通过双分子接触暂时形成。这些状态可以特别兴奋和检测与红色偏移波长，是稀疏和短寿命足以SMLM。通过调整 BODIPY 单体与激光参数的浓度，可实现本地化和噪声到噪声的最佳密度。在喂养和禁食条件下，我们解决了脂肪酸…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本出版物中报告的研究得到了国家卫生研究院国家普通医学研究所的支持，编号为R21GM127965。
Materials
| BODIPY C12 | ThermoFisher | D3822 | Green fatty acid analog |
| BODIPY C12 Red | ThermoFisher | D3835 | Red fatty acid analog |
| BODIPY(493/503) | ThermoFisher | D3922 | Neutral lipid marker |
| Concanavalin A | Sigma-Aldrich | C2010 | Cell immobilization on glass surface |
| Drop-out Mix Complete w/o nitrogen base | US Biological | D9515 | Amino acids for SCD |
| Dextrose | Sigma-Aldrich | G7021 | Carbon source for SCD |
| Eight Well | Cellvis | C8-1.58-N | Chambered Coverglasses |
| Eight Well, Lb-Tek II | Sigma-Aldrich | Chambered Coverglasses | |
| ET525/50 | Chroma | Bandpass filter | |
| ET595/50 | Chroma | Bandpass filter | |
| ET610/75 | Chroma | Bandpass filter | |
| Fetal Bovine Serum (FBS) | Gibco | 26140079 | Serum |
| FF652 | Semrock | Beam splitter | |
| FF731/137 | Semrock | Bandpass filter | |
| FluoroBrite DMEM | ThermoFisher | A1896701 | Cell culture medium |
| Hal4000 | Zhuang Lab, Harvard University | Data acquisition software | |
| Ixon89Ultra DU-897U | Andor | EMCCD camera for photon detection | |
| Laser 405, 488, 561, 640 nm | CW-OBIS | Lasers for excitation | |
| Insight3 | Zhuang Lab, Harvard University | Single molecule localization software | |
| L-Glutamine | Gibco | 25030-081 | Amino acid required for cell culture |
| live-cell imaging solution | ThermoFisher | A14291DJ | Imaging buffer |
| Lysotracker Green | ThermoFisher | L7526 | Bodipy based lysosome marker |
| Mammalian ATCC U2OS cells (Manassas, VA) | Dr. Jochen Mueller (University of Minnesota) | ||
| Nikon-CFI Apo 100 1.49 N.A | Nikon | Oil immersion objective | |
| Penicillin streptomycin | Gibco | 15140-122 | Antibiotics |
| Sodium Pyruvate | Gibco | 11360-070 | Supplement for cell culture |
| T562lpxr | Chroma | Beam splitter | |
| Trypsin-EDTA | Gibco | 15400-054 | Dissociation of adherent cell |
| W303 MATa strain | Horizon-Dharmacon | YSC1058 | Parental yeast strain |
| Yeast Nitrogen Base | Sigma-Aldrich | Y1250 | Nitrogen base without amino-acids |
| zt405/488/561/640rdc | Chroma | Quadband dichroic mirror | |
References
- Rust, M. J., Bates, M., Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods. 3 (10), 793-796 (2006).
- Betzig, E., et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 313 (5793), 1642-1645 (2006).
- Manley, S., et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Methods. 5 (2), 155-157 (2008).
- Wu, C. -. Y., Roybal, K. T., Puchner, E. M., Onuffer, J., Lim, W. A. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science. 350 (6258), 4077 (2015).
- Heilemann, M., et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angewandte Chemie. 47 (33), 6172-6176 (2008).
- Cordes, T., et al. Resolving single-molecule assembled patterns with superresolution blink-microscopy. Nano Letters. 10 (2), 645-651 (2010).
- Smith, E. M., Gautier, A., Puchner, E. M. Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System. ACS chemical biology. 14 (6), 1115-1120 (2019).
- Yan, Q., et al. Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins. Chemphyschem: A: European Journal of Chemical Physics and Physical Chemistry. 15 (4), 687-695 (2014).
- Jungmann, R., et al. Quantitative super-resolution imaging with qPAINT. Nature Methods. 13 (5), 439-442 (2016).
- Adhikari, S., Moscatelli, J., Smith, E. M., Banerjee, C., Puchner, E. M. Single-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells. Nature Communications. 10 (1), 1-12 (2019).
- Bergström, F., Mikhalyov, I., Hägglöf, P., Wortmann, R., Ny, T., Johansson, L. B. A. Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. Journal of the American Chemical Society. 124 (2), 196-204 (2002).
- Bröring, M., et al. Bis(BF2)-2,2′-bidipyrrins (BisBODIPYs): highly fluorescent BODIPY dimers with large stokes shifts. Chemistry (Weinheim an Der Bergstrasse, Germany). 14 (10), 2976-2983 (2008).
- Mikhalyov, I., Gretskaya, N., Bergström, F., Johansson, L. Electronic ground and excited state properties of dipyrrometheneboron difluoride (BODIPY): Dimers with application to biosciences. Physical Chemistry Chemical Physics. 4 (22), 5663-5670 (2002).
- Pagano, R. E., Chen, C. S. Use of BODIPY-labeled sphingolipids to study membrane traffic along the endocytic pathway. Annals of the New York Academy of Sciences. 845, 152-160 (1998).
- Bergström, F., Hägglöf, P., Karolin, J., Ny, T., Johansson, L. B. The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins. Proceedings of the National Academy of Sciences of the United States of America. 96 (22), 12477-12481 (1999).
- Kowada, T., Maeda, H., Kikuchi, K. BODIPY-based probes for the fluorescence imaging of biomolecules in living cells. Chemical Society Reviews. 44 (14), 4953-4972 (2015).
- Rocha, J. M., Gahlmann, A. Single-Molecule Tracking Microscopy – A Tool for Determining the Diffusive States of Cytosolic Molecules. Journal of Visualized Experiments: JoVE. (151), (2019).
- Sage, D., et al. Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software. Nature Methods. 16 (5), 387-395 (2019).
- Ovesný, M., Křížek, P., Borkovec, J., Svindrych, Z., Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics. 30 (16), 2389-2390 (2014).
- Puchner, E. M., Walter, J. M., Kasper, R., Huang, B., Lim, W. A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory. Proceedings of the National Academy of Sciences of the United States of America. 110 (40), 16015-16020 (2013).
- Shim, S. -. H., et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proceedings of the National Academy of Sciences. 109 (35), 13978-13983 (2012).
- Hansen, A. S., Woringer, M., Grimm, J. B., Lavis, L. D., Tjian, R., Darzacq, X. Robust model-based analysis of single-particle tracking experiments with Spot-On. eLife. 7, (2018).
- Bittel, A. M., Saldivar, I. S., Dolman, N. J., Nan, X., Gibbs, S. L. Superresolution microscopy with novel BODIPY-based fluorophores. PLoS ONE. 13 (10), (2018).
- Wijesooriya, C. S., Peterson, J. A., Shrestha, P., Gehrmann, E. J., Winter, A. H., Smith, E. A. A Photoactivatable BODIPY Probe for Localization-Based Super-Resolution Cellular Imaging. Angewandte Chemie (International Ed. in English). 57 (39), 12685-12689 (2018).
- Laissue, P. P., Alghamdi, R. A., Tomancak, P., Reynaud, E. G., Shroff, H. Assessing phototoxicity in live fluorescence imaging. Nature Methods. 14 (7), (2017).
- Wäldchen, S., Lehmann, J., Klein, T., van de Linde, S., Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Scientific Reports. 5, 15348 (2015).
- Grimm, J. B., et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nature methods. 14 (10), 987-994 (2017).
