Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Bioengineering
우리는 이광자 여기 형광 현미경(2PEF)과 통합된 산화중수소(중수D2O) 탐침 자극 라만 산란(DO-SRS) 현미경을 사용하여 아미노산에 의해 조절되는 세포에서 대사 활동을 직접 시각화하는 프로토콜을 제시합니다.
필수 방향족 아미노산(AAA)은 세포에서 새로운 바이오매스를 합성하고 정상적인 생물학적 기능을 유지하기 위한 빌딩 블록입니다. 예를 들어, AAA의 풍부한 공급은 암세포가 빠른 성장과 분열을 유지하는 데 중요합니다. 이를 통해 세포가 현장에서 대사를 위해 AAA를 활용하는 방법을 직접 시각화하기 위해 최소한의 샘플 준비로 매우 특이적이고 비침습적인 이미징 접근 방식에 대한 수요가 증가하고 있습니다. 여기에서 우리는 산화중수소(D2O) 프로빙과 유도 라만 산란(DO-SRS)을 결합하고 DO-SRS와 이광자 여기 형광(2PEF)을 단일 현미경에 통합하여 AAA 조절 하에 HeLa 세포의 대사 활동을 직접 시각화하는 광학 이미징 플랫폼을 개발합니다. 종합적으로, DO-SRS 플랫폼은 단일 HeLa 세포 단위에서 새로 합성된 단백질과 지질의 높은 공간 분해능과 특이성을 제공합니다. 또한, 2PEF 양식은 니코틴아미드 아데닌 디뉴클레오티드(NADH) 및 플라빈의 자가형광 신호를 무표지 방식으로 검출할 수 있습니다. 여기에 설명된 이미징 시스템은 시험관 내 및 생체 내 모델 모두와 호환되며, 이는 다양한 실험에 유연합니다. 이 프로토콜의 일반적인 워크플로에는 세포 배양, 배양 배지 준비, 세포 동기화, 세포 고정, DO-SRS 및 2PEF 양식을 사용한 샘플 이미징이 포함됩니다.
필수 방향족 아미노산인 페닐알라닌(Phe)과 트립토판(Tryp)은 인체에 흡수되어 정상적인 생물학적 기능을 유지하기 위한 새로운 분자를 합성할 수 있습니다1. Phe는 단백질, 멜라닌 및 티로신의 합성에 필요하고 Tryp은 멜라토닌, 세로토닌 및 니아신 2,3의 합성에 필요합니다. 그러나 이러한 AAA의 과도한 섭취는 포유류의 라파마이신(mTOR) 경로 표적을 상향 조절하고, AMP 활성화 단백질 키나아제를 억제하며, 미토콘드리아 대사를 방해하여 거대분자 생합성을 집합적으로 변화시키고 건강한 세포에서 활성 산소종(ROS)과 같은 악성 전구체의 생성을 유도할 수 있습니다 4,5,6. 과도한 AAA 조절 하에서 변화된 대사 역학의 직접적인 시각화는 암 발병과 건강한 세포의 성장을 촉진하는 AAA의 역할을 이해하는 데 필수적입니다 7,8,9.
1. 매체 준비
50%D2O-함유 세포 배양 배지에 15x 농도의 과량의 AAA를 첨가하면 HeLa 세포에서 새로 합성된 지질 및 단백질의 뚜렷한 C-D 라만 밴드가 생성되었습니다(그림 2B). 이전 실험은 2x 및 5x와 같은 다양한 농도 수준으로 수행되었으며 데이터가 제시되지는 않았지만 15x 농도는 새로 합성된 지질 및 단백질의 가장 뚜렷한 C-D 라만 밴드를 생성했습니다. 구체적으로, 지질 방울(LD)을 .......
Name | Company | Catalog Number | Comments |
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-R980-25x36 | 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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