Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Bioengineering
Vi presenterer en protokoll for direkte å visualisere metabolske aktiviteter i celler regulert av aminosyrer ved bruk av deuteriumoksid (tungtvann D2O) probed stimulert Raman-spredning (DO-SRS) mikroskopi, som er integrert med to-foton eksitasjonsfluorescensmikroskopi (2PEF).
Essensielle aromatiske aminosyrer (AAA) er byggesteiner for å syntetisere nye biomasser i celler og opprettholde normale biologiske funksjoner. For eksempel er en rikelig tilførsel av AAA viktig for kreftceller for å opprettholde sin raske vekst og deling. Med dette er det en økende etterspørsel etter en svært spesifikk, ikke-invasiv bildebehandling tilnærming med minimal prøveforberedelse for direkte å visualisere hvordan celler utnytter AAA for deres metabolisme in situ. Her utvikler vi en optisk bildebehandlingsplattform som kombinerer deuteriumoksid (D2O) sondering med stimulert Raman-spredning (DO-SRS) og integrerer DO-SRS med to-foton eksitasjonsfluorescens (2PEF) i et enkelt mikroskop for direkte å visualisere de metabolske aktivitetene til HeLa-celler under AAA-regulering. Samlet gir DO-SRS-plattformen høy romlig oppløsning og spesifisitet av nylig syntetiserte proteiner og lipider i enkelt HeLa-celleenheter. I tillegg kan 2PEF-modaliteten oppdage autofluorescenssignaler av nikotinamid-adenin-dinukleotid (NADH) og Flavin på en etikettfri måte. Avbildningssystemet beskrevet her er kompatibelt med både in vitro - og in vivo-modeller , som er fleksibelt for ulike eksperimenter. Den generelle arbeidsflyten til denne protokollen inkluderer cellekultur, forberedelse av kulturmedier, cellesynkronisering, cellefiksering og prøveavbildning med DO-SRS- og 2PEF-modaliteter.
Å være essensielle aromatiske aminosyrer (AAA), fenylalanin (Phe) og tryptofan (Tryp) kan absorberes av menneskekroppen for å syntetisere nye molekyler for å opprettholde normale biologiske funksjoner1. Phe er nødvendig for syntese av proteiner, melanin og tyrosin, mens Tryp er nødvendig for syntesen av melatonin, serotonin og niacin 2,3. Imidlertid kan overskytende forbruk av disse AAAene oppregulere pattedyrmålet for rapamycin (mTOR) -banen, hemme AMP-aktivert proteinkinase og forstyrre mitokondriell metabolisme, kollektivt endre makromolekylbiosyntese og føre til produksjon av ondart....
1. Media forberedelse
Tilsetningen av overskytende AAA ved 15x konsentrasjoner til 50% D2O-holdige cellekulturmedier produserte distinkte C-D Raman-bånd av nylig syntetiserte lipider og proteiner i HeLa-celler (figur 2B). Tidligere eksperimenter ble utført med forskjellige konsentrasjonsnivåer, for eksempel 2x og 5x, og selv om dataene ikke presenteres, produserte 15x-konsentrasjonen de mest distinkte C-D Raman-båndene av nylig syntetiserte lipider og proteiner. Spesielt, ved å undersøke lipiddr?.......
DO-SRS og 2PEF-avbildning har blitt brukt til å undersøke metabolsk dynamikk i ulike ex vivo-modeller, inkludert Drosophila og humant vev 21,22,23,24,26,27,33. Avbildningsmodaliteten som brukes i denne studien integrerer DO-SRS og 2PEF-mikroskopi, som kan overgå andre molekylspes.......
Vi takker Dr. Yajuan Li og Anthony Fung for deres tekniske støtte, og Fraley-laboratoriet for cellelinjen. Vi anerkjenner oppstartsmidlene fra UCSD, NIH U54CA132378, NIH 5R01NS111039, NIH R21NS125395, NIHU54DK134301, NIHU54 HL165443 og Hellman Fellow Award.
....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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