芳香族氨基酸调控细胞代谢的生物正交化学成像
Summary
我们提出了一种协议，使用氧化氘（重水D2O）探测的受激拉曼散射（DO-SRS）显微镜直接可视化由氨基酸调节的细胞中的代谢活动，该显微镜与双光子激发荧光显微镜（2PEF）集成。
Abstract
必需芳香族氨基酸（AAA）是在细胞中合成新生物质和维持正常生物功能的基石。例如，充足的AAAs供应对于癌细胞维持其快速生长和分裂很重要。因此，对高度特异性、无创成像方法的需求不断增长，该方法具有最少的样品制备，以直接可视化细胞如何利用 AAA 进行 原位代谢。在这里，我们开发了一种光学成像平台，该平台将氧化氘（D2O）探测与受激拉曼散射（DO-SRS）相结合，并将DO-SRS与双光子激发荧光（2PEF）集成到单个显微镜中，以直接可视化AAA调节下HeLa细胞的代谢活动。总的来说，DO-SRS平台为单个HeLa细胞单元中新合成的蛋白质和脂质提供了高空间分辨率和特异性。此外，2PEF模式可以无标记方式检测烟酰胺腺嘌呤二核苷酸（NADH）和黄素的自发荧光信号。这里描述的成像系统与 体外 和 体内 模型兼容，对于各种实验都是灵活的。该协议的一般工作流程包括细胞培养、培养基制备、细胞同步、细胞固定以及使用 DO-SRS 和 2PEF 模式进行样品成像。
Introduction
苯丙氨酸（Phe）和色氨酸（Tryp）是必需的芳香族氨基酸（AAA），可以被人体吸收，合成维持正常生物学功能的新分子1。Phe是合成蛋白质，黑色素和酪氨酸所必需的，而Tryp是合成褪黑激素，血清素和烟酸2,3所必需的。然而，过量食用这些AAA可以上调雷帕霉素（mTOR）途径的哺乳动物靶标，抑制AMP活化的蛋白激酶，并干扰线粒体代谢，共同改变大分子生物合成并导致恶性前体的产生，例如健康细胞中的活性氧（ROS）4,5,6.在过量的AAA调节下直接可视化改变的代谢动力学对于了解AAA在促进癌症发展和健康细胞生长中的作用至关重要7,8,9。
传统的AAA研究依赖于气相色谱（GC）10。其他方法，如磁共振成像（MRI），空间分辨率有限，因此难以对生物样品进行细胞和亚细胞分析11。最近，基质辅助激光解吸/电离（MALDI）已被开发出来，以阐明AAA在脂质和蛋白质合成中的作用，使用非侵入性生物标志物12,13,14在癌症增殖中的作用。然而，这种技术仍然存在成像深度浅、空间分辨率差和样品制备量大等问题。在细胞水平上，无毒稳定同位素，如氮-15和碳-13，可以通过多同位素成像和纳米级二次离子质谱法进行追踪，以了解它们掺入大分子中。然而，这些方法对活的生物样品15,16具有破坏性。原子力显微镜（AFM）是另一种强大的技术，可以可视化代谢动力学17。另一方面，AFM成像期间的缓慢扫描速率可能会导致热漂移导致结果的图像失真。
我们通过耦合氧化氘（D2O）探测的受激拉曼散射（DO-SRS）显微镜和无标记双光子激发荧光显微镜（2PEF）来开发一种无创双正交成像模式。这种模式在对生物样品18,19,20,21,22,23,24进行成像时实现了高空间分辨率和化学特异性。该协议介绍了DO-SRS和2PEF的应用，以检查癌症进展过程中脂质，蛋白质和氧化还原比率变化的代谢动力学。由于D2O是水的稳定同位素形式，细胞生物分子可以用氘（D）标记，因为它可以快速补偿细胞中的全身水分，通过酶交换形成碳 – 氘（C-D）键21。新合成的大分子（包括脂质、蛋白质、DNA/RNA 和碳水化合物）中的 C-D 键可以在拉曼光谱20、21、22、25、26、27 的细胞静默区域中检测到。通过两个同步激光脉冲，新合成的脂质和蛋白质的C-D键可以通过高光谱成像（HSI）显示在单个细胞上，而无需用细胞毒性剂提取或标记它们。此外，SRS显微镜能够通过捕获和组合一组横截面图像来构建生物样品中选定感兴趣区域的三维（3D）模型22,26。通过高光谱和3D体积成像，DO-SRS可以获得单细胞中新合成的大分子的空间分布，以及根据AAA法规22促进癌症生长过程的细胞器类型。此外，使用2PEF，我们可以获得黄素和烟酰胺腺嘌呤二核苷酸（NADH）的自发荧光信号，具有高分辨率，深度穿透深度和生物样品中的低水平损伤21,23,24。黄素和NADH自发荧光信号已被用于表征癌细胞中的氧化还原稳态和脂质过氧化22,26。因此，DO-SRS和2PEF的偶联不仅提供了癌细胞中AAA调节代谢动力学的亚细胞分析，具有高空间分布，化学特异性信息和最少的样品制备，而且该方法还减少了用有毒试剂提取或标记内源性分子的需要。在该协议中，我们首先介绍了D2O和氨基酸制备以及癌细胞培养的程序。然后，我们展示了DO-SRS成像和2PEF成像的协议。最后，我们介绍了SRS和2PEF成像的代表性结果，证明了AAA调节的脂质和蛋白质代谢变化以及癌细胞中的氧化还原比例变化。图 1 突出显示了该过程的详细图示。
Protocol
1. 培养基制备 在含有 50% D2O 的 Dulbecco 改良鹰培养基 （DMEM） 中制备 10 mL 对照和过量 AAA。对于对照介质，在 15 mL 锥形管中测量 10 mg DMEM 粉末与 4.7 mL 双蒸水 （ddH2O） 并混合。DMEM粉末含有标准浓度的所有氨基酸。彻底涡旋并倒置管，以确保溶液充分混合。加入 4.7 mL D2O、0.5 mL 胎牛血清 （5% FBS） 和 0.1 mL 青霉素/链霉素 （1%）。彻底涡旋并倒置管，…
Representative Results
将浓度为 15x 的过量 AAA 添加到含 50% D2O 的细胞培养基中，在 HeLa 细胞中产生了新合成的脂质和蛋白质的不同 C-D 拉曼带（图 2B）。以前的实验是用不同的浓度水平进行的，例如2x和5x，虽然没有提供数据，但15x浓度产生了新合成的脂质和蛋白质的最明显的C-D拉曼带。具体来说，通过研究脂滴（LDs），我们注意到15x Phe和15x Tryp分别在2,143 cm-1和2,172 cm-1处诱导新合…
Discussion
DO-SRS和2PEF成像已被应用于研究各种离体模型中的代谢动力学，包括果蝇和人体组织21,22,23,24,26,27,33。本研究中使用的成像方式集成了DO-SRS和2PEF显微镜，无需使用细胞毒性试剂进行分子提取或标记，并且需要最少的样…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢李亚娟博士和冯安东尼博士的技术支持，以及Fraley实验室的细胞系。我们感谢UCSD，NIH U54CA132378，NIH 5R01NS111039，NIH R21NS125395，NIHU54DK134301，NIHU54 HL165443和Hellman Fellow奖的启动资金。
Materials
| 10 mL Serological Pipettes | Avantor (by VWR) | 75816-100 | https://us.vwr.com/store/product?keyword=75816-100 |
| 15 mL Conical Centrifuge Tube | VWR | 89039-664 | https://mms.mckesson.com/product/1001859/VWR-International-89039-664 |
| 16% Formaldehyde, Methanol-free | ThermoFisher Scientific | 28906 | https://www.thermofisher.com/order/catalog/product/28906 |
| 24-well plate | Fisherbrand | FB0112929 | https://www.fishersci.com/shop/products/24-well-tc-multidish-100-cs/FB012929#?keyword=FB012929 |
| 25 mm Syringe Filter, 2 μm PES | Foxx Life Sciences | 381-2216-OEM | https://www.foxxlifesciences.com/collections/pes-syringe-filters/products/381-2216-oem?variant=16274336003 |
| 460 nm Filter Cube | Olympus | OCT-ET460/50M32 | |
| AC Adapters of the Power Supply for LD OBIS 6 Laser Remote | Olympus | Supply power to the laser | |
| Band-pass Filter | KR Electronics | KR2724 | 8 MHz |
| BNC 50 Ohm Terminator | Mini Circuits | STRM-50 | |
| BNC Cable | Thorlabs | 2249-C | Coaxial Cable, BNC Male/Male |
| Broadband Dielectric Mirror | Thorlabs | BB1-E03 | 750 – 1100 nm |
| Centrifuge | |||
| Condenser | Olympus | ||
| Cover Glass | Corning | 2850-25 | https://ecatalog.corning.com/life-sciences/b2b/NL/en/Glassware/Cover-Glass/Corning%C2%AE-Square-%231%C2%BD-Cover-Glass/p/2850-25 |
| DC power supply | TopWard | 6302D | |
| Dichroic Mount | Thorlabs | KM100CL | |
| Dimethyl Sulfoxide Cell Culture Reagent | mpbio | 196055 | https://www.mpbio.com/0219605525-dimethyl-sulfoxide-cf |
| Dulbecco's Modified Eagle’s Medium without Methionine, Threonine, and Sodium Pyruvate | MilliporeSigma | 38210000 | https://www.usbio.net/media/D9800-22/dulbeccorsquos-mem-dmem-wsodium-bicarbonate-wo-methionine-threonine-sodium-pyruvate-powder With Sodium Bicarbonate and without Methionine, Threonine, and Sodium Pyruvate |
| Dulbecco’s Modified Eagle’s Medium | Corning | MT10027CV | https://www.fishersci.com/shop/products/dmem-dulbecco-s-modified-eagle-s-medium-4/MT10027CV#:~:text=Dulbecco's%20Modified%20Eagle's%20Medium%20 |
| FIJI ImageJ | ImageJ | Version 1.53t 24 August 2022 | https://imagej.net/software/fiji/downloads |
| Heavy Water (Deuterium Oxide) | Cambridge Isotope Laboratories, Inc. | 7732-18-5 | https://shop.isotope.com/productdetails.aspx?itemno=DLM-4-1L |
| Hela Cells | ATCC | CCL-2 | https://www.atcc.org/products/ccl-2 |
| Hemocymeter | MilliporeSigma | Z359629-1EA | https://www.sigmaaldrich.com/US/en/product/sigma/z359629?gclid=Cj0KCQiA37KbBhDgARIsAI zce15A5FIy0WS7I6ec2KVk QPXVMEqlAnYis_bKB6P6lr SIZ-wAXOyAELIaAhhEEAL w_wcB&gclsrc=aw.ds |
| High O.D. Bandpass Filter | Chroma Technology | ET890/220m | Filter the Stokes beam and transmit the pump beam |
| HyClone Fetal Bovine Serum (FBS) | Cytiva | SH300880340 | https://www.fishersci.com/shop/products/hyclone-fetal-bovine-serum-u-s-standard-4/SH300880340 |
| HyClone Trypsin 0.25% (1x) Solution | Cytiva | SH30042.02 | https://www.cytivalifesciences.com/en/us/shop/cell-culture-and-fermentation/reagents-and-supplements/cell-disassociation-reagents/hyclone-trypsin-protease-p-00445 |
| Integrated SRS Laser System | Applied Physics & Electronics, Inc. | picoEMERALD | picoEMERALD provides an output pulse at 1031 nm with 6-ps pulse width and 80-MHz repetition rate, which serves as the Stokes beam. The frequency doubled beam at 532 nm is used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with five~6 ps pulse width (the idler beam of the OPO is blocked with an,interferometric filter). The output wavelength of the OPO is tunable from 720–950 nm, which serves as the pump beam. The intensity of the 1031 nm Stokes beam is modulated sinusoidally by a built-in EOM at 8 MHz with a modulation depth of more than 90%. The pump beam is spatially overlapped with the Stokes beam by using a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulses are achieved with a built-in delay stage and optimized by the SRS signal of pure D2O at the microscope. |
| Inverted Laser-scanning Microscope | Olympus | FV1200MPE | |
| IX3-CBH Control box | Olympus | Control the laser-scanning microscope | |
| Kinematic Mirror Mount | Thorlabs | POLARIS-K1-2AH | 2 Low-Profile Hex Adjusters |
| L-Phenalynine | Sigma | P5482-25G | https://www.sigmaaldrich.com/US/en/product/sigma/p5482 |
| L-Tryptophan | Sigma | T8941-25G | https://www.sigmaaldrich.com/US/en/product/sigma/t8941 |
| LabSpec 6 | Horiba XploRA | N/A | https://www.horiba.com/gbr/scientific/products/detail/action/show/Product/labspec-6-spectroscopy-suite-software-1843/ |
| Lock-In Amplifier | Zurich Instruments | N/A | https://www.zhinst.com/americas/en/products/shfli-lock-in-amplifier |
| Long-pass Dichroic Beam Splitter | Semrock | Di02-R980-25×36 | 980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter |
| MATLAB | MathWorks | Version: R2022b | https://www.mathworks.com/products/new_products/latest_features.html |
| Microscope Slides | Fisherbrand | 12-550-003 | https://www.fishersci.com/shop/products/fisherbrand-selectfrost-microscope-slides-9/12550003#?keyword=12-550-003 |
| Microscopy Imaging Software | Olympus | FluoView | |
| MPLN 100x, Olympus | Olympus | MPLAPON | https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11364 |
| MPLN 50x, Olympus | Olympus | MPLAPON | https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11363 |
| NA Oil Condenser | Olympus | 6-U130 | https://www.hitechinstruments.com/Product-Details/olympus-achromatic-aplanatic-high-na-condneser |
| Nail Polish | Wet n Wild | B01EO2G5O4 | https://www.amazon.com/dp/B01EO2G5O4/ref=cm_sw_r_api_i_E609VVDWW HHQP38FXXDC_0 |
| Origin | OriginLab | Origin 2022b (9.95) | https://www.originlab.com/index.aspx?go=PRODUCTS/Origin |
| Parafilm | Fisher Scientific | S37440 | https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/p-2379782 |
| PBS 1x (Dulbecco's Phosphate Buffered Saline) | Thermofischer – Gibco | 14040117 | https://www.thermofisher.com/order/catalog/product/14040117?SID=srch-hj-14040117 |
| Penicillin/Streptomycin | Thermofischer – Gibco | 15140122 | https://www.thermofisher.com/order/catalog/product/15140122 |
| Periscope Assembly | Thorlabs | RS99 | Includes the top and bottom units, Ø1" post, and clamping fork. |
| picoEmerald System | A.P.E | N/A | https://www.ape-berlin.de/en/cars-srs/ |
| Shielded Box with BNC Connectors | Pomona Electronics | 2902 | Aluminum Box with Cover, BNC Female/Female |
| Si Photodiode Detector | Home Built | N/A | DYI series |
| Silicon Wafer | |||
| Spacers | Grace Bio-Labs | 654008 | https://gracebio.com/product/secureseal-imaging-spacers-654008/ |
| Spontaneous Raman spectroscopy | Horiba XploRA | N/A | https://www.horiba.com/int/products/detail/action/show/Product/xploratm-plus-1528/ |
| Stimulated Raman Scattering Microscopy | Home Built | N/A | |
| Touch Panel Controller | Olympus | Control the X-Y direction of the laser-scanning microscope | |
| Trypan Blue 0.4% (0.85% NaCl) | Lonza | 17-942E | https://bioscience.lonza.com/lonza_bs/US/en/Culture-Media-and-Reagents/p/000000000000181876/Trypan-Blue%2C-0-4%25-Solution" |
| Tweezers | Kaverme – Amazon | B07RNVXXV1 | https://www.amazon.com/Precision-Anti-Static-Electronics-Laboratory-Jewelry-Making/dp/B07RNVXXV1" |
| Two Photon Excitation Fluorescence Microscopy | Home Built | N/A | |
| Weighing Paper | VWR | 12578-165 | https://us.vwr.com/store/product/4597617/vwr-weighing-paper |
| Zurich LabOneQ Software | Zurich Instruments | Control the Zurich lock-in amplifier |
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Cite This Article
Bagheri, P., Hoang, K., Kuo, C., Trivedi, H., Jang, H., Shi, L. Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids. J. Vis. Exp. (195), e65121, doi:10.3791/65121 (2023).