JoVE Logo
Faculty Resource Center
Authors
Librarians
High Schools
About

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Aromatik Amino Asitler Tarafından Düzenlenen Hücre Metabolizmasının Biyoortogonal Kimyasal Görüntülenmesi

Published: May 12th, 2023

DOI:

10.3791/65121

1Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego
* These authors contributed equally

İki foton uyarma floresan mikroskobu (2PEF) ile entegre olan döteryum-oksit (ağır suD2O) problu uyarılmış Raman saçılımı (DO-SRS) mikroskobu kullanılarak amino asitler tarafından düzenlenen hücrelerdeki metabolik aktiviteleri doğrudan görselleştirmek için bir protokol sunuyoruz.

Esansiyel aromatik amino asitler (AAA'lar), hücrelerde yeni biyokütlelerin sentezlenmesi ve normal biyolojik fonksiyonların sürdürülmesi için yapı taşlarıdır. Örneğin, kanser hücrelerinin hızlı büyümelerini ve bölünmelerini sürdürmeleri için bol miktarda AAA kaynağı önemlidir. Bununla birlikte, hücrelerin yerinde metabolizmaları için AAA'lardan nasıl yararlandığını doğrudan görselleştirmek için minimum numune hazırlığı ile son derece spesifik, noninvaziv bir görüntüleme yaklaşımına yönelik artan bir talep vardır. Burada, döteryum oksit (D2O) sondalamasını uyarılmış Raman saçılımı (DO-SRS) ile birleştiren ve DO-SRS'yi iki fotonlu uyarma floresansı (2PEF) ile entegre eden bir optik görüntüleme platformu geliştiriyoruz. Toplu olarak, DO-SRS platformu, tek HeLa hücre birimlerinde yeni sentezlenen proteinlerin ve lipitlerin yüksek uzamsal çözünürlüğünü ve özgüllüğünü sağlar. Ek olarak, 2PEF modalitesi, nikotinamid adenin dinükleotidi (NADH) ve Flavin'in otofloresan sinyallerini etiketsiz bir şekilde tespit edebilir. Burada açıklanan görüntüleme sistemi, çeşitli deneyler için esnek olan hem in vitro hem de in vivo modellerle uyumludur. Bu protokolün genel iş akışı, hücre kültürü, kültür ortamı hazırlama, hücre senkronizasyonu, hücre fiksasyonu ve DO-SRS ve 2PEF modaliteleri ile numune görüntülemeyi içerir.

Esansiyel aromatik amino asitler (AAA'lar), fenilalanin (Phe) ve triptofan (Trip), normal biyolojik fonksiyonları sürdürmek için yeni moleküller sentezlemek üzere insan vücudu tarafından emilebilir1. Fenilalan, proteinler, melanin ve tirozin sentezi için gereklidir, Tryp ise melatonin, serotonin ve niasin 2,3 sentezi için gereklidir. Bununla birlikte, bu AAA'ların aşırı tüketimi, rapamisin (mTOR) yolunun memeli hedefini yukarı regüle edebilir, AMP ile aktive olan protein kinazını inhibe edebilir ve mitokondriyal metabolizmaya müdahale ederek makromolekül biyosentezini toplu olarak değiş....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Medya hazırlığı

  1. Dulbecco'nun %50D2O içeren modifiye Eagle besiyerinde (DMEM) 10 mL kontrol ve fazla AAA hazırlayın.
    1. Kontrol ortamı için, 15 mL'lik konik bir tüpte 10 mg DMEM tozunu 4.7 mL çift damıtılmış su (ddH2O) ile ölçün ve karıştırın. DMEM tozu, standart konsantrasyonlarda tüm amino asitleri içerir. Çözeltinin iyice karıştığından emin olmak için tüpü iyice girdap yapın ve ters çevirin. 4.7 mLD2O, 0.5 mL fetal s?.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

%50D2O içeren hücre kültürü ortamına 15x konsantrasyonda fazla AAA'ların eklenmesi, HeLa hücrelerinde yeni sentezlenen lipitlerin ve proteinlerin farklı C-D Raman bantlarını üretti (Şekil 2B). Önceki deneyler, 2x ve 5x gibi farklı konsantrasyon seviyelerinde gerçekleştirildi ve veriler sunulmamasına rağmen, 15x konsantrasyonu, yeni sentezlenen lipitlerin ve proteinlerin en belirgin C-D Raman bantlarını üretti. Spesifik olarak, lipid damlacıklarını (LD'ler).......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

DO-SRS ve 2PEF görüntüleme, Drosophila ve insan dokuları 21,22,23,24,26,27,33 dahil olmak üzere çeşitli ex vivo modellerde metabolik dinamikleri araştırmak için uygulanmıştır. Bu çalışmada kullanılan görüntüleme yöntemi, molekül ekstraksiyonu veya sitotoksik re.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Teknik destekleri için Dr. Yajuan Li ve Anthony Fung'a ve hücre hattı için Fraley laboratuvarına teşekkür ederiz. UCSD, NIH U54CA132378, NIH 5R01NS111039, NIH R21NS125395, NIHU54DK134301, NIHU54 HL165443 ve Hellman Fellow Award'dan gelen başlangıç fonlarını kabul ediyoruz.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
10 mL Serological Pipettes Avantor (by VWR)75816-100https://us.vwr.com/store/product?keyword=75816-100
15 mL Conical Centrifuge TubeVWR89039-664https://mms.mckesson.com/product/1001859/VWR-International-89039-664
16% Formaldehyde, Methanol-freeThermoFisher Scientific28906https://www.thermofisher.com/order/catalog/product/28906
24-well plateFisherbrandFB0112929https://www.fishersci.com/shop/products/24-well-tc-multidish-100-cs/FB012929#?keyword=FB012929
25 mm Syringe Filter, 2 μm PESFoxx Life Sciences381-2216-OEMhttps://www.foxxlifesciences.com/collections/pes-syringe-filters/products/381-2216-oem?variant=16274336003
460 nm Filter CubeOlympusOCT-ET460/50M32
AC Adapters of the Power Supply for LD OBIS 6 Laser RemoteOlympusSupply power to the laser
Band-pass FilterKR ElectronicsKR27248 MHz
BNC 50 Ohm Terminator Mini CircuitsSTRM-50
BNC CableThorlabs2249-CCoaxial Cable, BNC Male/Male
Broadband Dielectric MirrorThorlabsBB1-E03750 - 1100 nm
Centrifuge
CondenserOlympus
Cover GlassCorning2850-25https://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 supplyTopWard6302D
Dichroic MountThorlabsKM100CL
Dimethyl Sulfoxide Cell Culture Reagentmpbio 196055https://www.mpbio.com/0219605525-dimethyl-sulfoxide-cf
Dulbecco's Modified Eagle’s Medium without Methionine, Threonine, and Sodium PyruvateMilliporeSigma38210000https://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 MediumCorningMT10027CVhttps://www.fishersci.com/shop/products/dmem-dulbecco-s-modified-eagle-s-medium-4/MT10027CV#:~:text=Dulbecco's%20Modified%20Eagle's%20Medium%20
FIJI ImageJImageJVersion 1.53t 24 August 2022https://imagej.net/software/fiji/downloads
Heavy Water (Deuterium Oxide)Cambridge Isotope Laboratories, Inc.7732-18-5https://shop.isotope.com/productdetails.aspx?itemno=DLM-4-1L
Hela CellsATCCCCL-2https://www.atcc.org/products/ccl-2
HemocymeterMilliporeSigmaZ359629-1EAhttps://www.sigmaaldrich.com/US/en/product/sigma/z359629?gclid=Cj0KCQiA37KbBhDgARIsAI
zce15A5FIy0WS7I6ec2KVk
QPXVMEqlAnYis_bKB6P6lr
SIZ-wAXOyAELIaAhhEEAL
w_wcB&gclsrc=aw.ds
High O.D. Bandpass FilterChroma TechnologyET890/220mFilter the Stokes beam and transmit the pump beam
HyClone Fetal Bovine Serum (FBS)Cytiva SH300880340https://www.fishersci.com/shop/products/hyclone-fetal-bovine-serum-u-s-standard-4/SH300880340
HyClone Trypsin 0.25% (1x) SolutionCytivaSH30042.02https://www.cytivalifesciences.com/en/us/shop/cell-culture-and-fermentation/reagents-and-supplements/cell-disassociation-reagents/hyclone-trypsin-protease-p-00445
Integrated SRS Laser SystemApplied Physics & Electronics, Inc.picoEMERALDpicoEMERALD 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 MicroscopeOlympusFV1200MPE
IX3-CBH Control boxOlympusControl the laser-scanning microscope
Kinematic Mirror MountThorlabsPOLARIS-K1-2AH2 Low-Profile Hex Adjusters
L-PhenalynineSigmaP5482-25Ghttps://www.sigmaaldrich.com/US/en/product/sigma/p5482
L-TryptophanSigmaT8941-25Ghttps://www.sigmaaldrich.com/US/en/product/sigma/t8941
LabSpec 6Horiba XploRAN/Ahttps://www.horiba.com/gbr/scientific/products/detail/action/show/Product/labspec-6-spectroscopy-suite-software-1843/
Lock-In AmplifierZurich InstrumentsN/Ahttps://www.zhinst.com/americas/en/products/shfli-lock-in-amplifier
Long-pass Dichroic Beam SplitterSemrockDi02-R980-25x36980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter
MATLABMathWorksVersion: R2022bhttps://www.mathworks.com/products/new_products/latest_features.html
Microscope SlidesFisherbrand12-550-003https://www.fishersci.com/shop/products/fisherbrand-selectfrost-microscope-slides-9/12550003#?keyword=12-550-003
Microscopy Imaging SoftwareOlympusFluoView
MPLN 100x, OlympusOlympusMPLAPONhttps://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11364
MPLN 50x, OlympusOlympusMPLAPON https://www.olympus-ims.com/en/microscope/mplapon/#!cms[focus]=cmsContent11363
NA Oil CondenserOlympus 6-U130https://www.hitechinstruments.com/Product-Details/olympus-achromatic-aplanatic-high-na-condneser
Nail PolishWet n WildB01EO2G5O4https://www.amazon.com/dp/B01EO2G5O4/ref=cm_sw_r_api_i_E609VVDWW
HHQP38FXXDC_0
OriginOriginLabOrigin 2022b (9.95)https://www.originlab.com/index.aspx?go=PRODUCTS/Origin
ParafilmFisher ScientificS37440https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/p-2379782
PBS 1x (Dulbecco's Phosphate Buffered Saline)Thermofischer - Gibco14040117https://www.thermofisher.com/order/catalog/product/14040117?SID=srch-hj-14040117
Penicillin/StreptomycinThermofischer - Gibco15140122https://www.thermofisher.com/order/catalog/product/15140122
Periscope AssemblyThorlabsRS99Includes the top and bottom units, Ø1" post, and clamping fork.
picoEmerald SystemA.P.EN/Ahttps://www.ape-berlin.de/en/cars-srs/
Shielded Box with BNC ConnectorsPomona Electronics2902Aluminum Box with Cover, BNC Female/Female
Si Photodiode DetectorHome BuiltN/ADYI series
Silicon Wafer
SpacersGrace Bio-Labs654008https://gracebio.com/product/secureseal-imaging-spacers-654008/
Spontaneous Raman spectroscopyHoriba XploRAN/Ahttps://www.horiba.com/int/products/detail/action/show/Product/xploratm-plus-1528/
Stimulated Raman Scattering MicroscopyHome BuiltN/A
Touch  Panel ControllerOlympusControl the X-Y direction of the laser-scanning microscope
Trypan Blue 0.4% (0.85% NaCl) Lonza17-942Ehttps://bioscience.lonza.com/lonza_bs/US/en/Culture-Media-and-Reagents/p/000000000000181876/Trypan-Blue%2C-0-4%25-Solution"
TweezersKaverme - AmazonB07RNVXXV1https://www.amazon.com/Precision-Anti-Static-Electronics-Laboratory-Jewelry-Making/dp/B07RNVXXV1"
Two Photon Excitation Fluorescence MicroscopyHome BuiltN/A
Weighing Paper VWR12578-165https://us.vwr.com/store/product/4597617/vwr-weighing-paper
Zurich LabOneQ SoftwareZurich InstrumentsControl the Zurich lock-in amplifier

  1. Wu, G. Functional amino acids in nutrition and health. Amino Acids. 45 (3), 407-411 (2013).
  2. Wei, Z., Liu, X., Cheng, C., Yu, W., Yi, P. Metabolism of amino acids in cancer. Frontiers in Cell and Developmental Biology. 8, 603837 (2020).
  3. Parthasarathy, A., et al. A three-ring circus: Metabolism of the three proteogenic aromatic amino acids and their role in the health of plants and animals. Frontiers in Molecular Biosciences. 5, 29 (2018).
  4. Wang, H., et al. l-tryptophan activates mammalian target of rapamycin and enhances expression of tight junction proteins in intestinal porcine epithelial cells. The Journal of Nutrition. 145 (6), 1156-1162 (2015).
  5. Saxton, R. A., Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell. 168 (6), 960-976 (2017).
  6. Mossmann, D., Park, S., Hall, M. N. mTOR signalling and cellular metabolism are mutual determinants in cancer. Nature Reviews. Cancer. 18 (12), 744-757 (2018).
  7. Kimura, T., Watanabe, Y. Tryptophan protects hepatocytes against reactive oxygen species-dependent cell death via multiple pathways including Nrf2-dependent gene induction. Amino Acids. 48 (5), 1263-1274 (2016).
  8. Ma, Q., et al. Dietary supplementation with aromatic amino acids decreased triglycerides and alleviated hepatic steatosis by stimulating bile acid synthesis in mice. Food and Function. 12 (1), 267-277 (2021).
  9. Cheng, C., et al. Treatment implications of natural compounds targeting lipid metabolism in nonalcoholic fatty liver disease, obesity and cancer. International Journal of Biological Sciences. 15 (8), 1654-1663 (2019).
  10. Lubes, G., Goodarzi, M. GC-MS based metabolomics used for the identification of cancer volatile organic compounds as biomarkers. Journal of Pharmaceutical and Biomedical Analysis. 147, 313-322 (2018).
  11. Di Gialleonardo, V., Wilson, D. M., Keshari, K. R. The potential of metabolic imaging. Seminars in Nuclear Medicine. 46 (1), 28-39 (2016).
  12. Bowman, A. P., et al. Evaluation of lipid coverage and high spatial resolution MALDI-imaging capabilities of oversampling combined with laser post-ionisation. Analytical and Bioanalytical Chemistry. 412 (10), 2277-2289 (2020).
  13. Murphy, R. C., Hankin, J. A., Barkley, R. M. Imaging of lipid species by MALDI mass spectrometry. Journal of Lipid Research. 50, 317-322 (2009).
  14. Pirman, D. A., et al. Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry. PLoS One. 8 (4), e61379 (2013).
  15. Li, Z., et al. Single-cell lipidomics with high structural specificity by mass spectrometry. Nature Communications. 12 (1), 2869 (2021).
  16. Miyagi, M., Kasumov, T. Monitoring the synthesis of biomolecules using mass spectrometry. Philosophical Transactions. Series A, Mathematical, Physical and Engineering Sciences. 374 (2079), 20150378 (2016).
  17. Wang, T., Shogomori, H., Hara, M., Yamada, T., Kobayashi, T. Nanomechanical recognition of sphingomyelin-rich membrane domains by atomic force microscopy. Biochemistry. 51 (1), 74-82 (2012).
  18. Fung, A. A., Shi, L. Mammalian cell and tissue imaging using Raman and coherent Raman microscopy. Wiley Interdisciplinary Reviews. Systems Biology and Medicine. 12 (6), e1501 (2020).
  19. Shi, L., Fung, A. A., Zhou, A. Advances in stimulated Raman scattering imaging for tissues and animals. Quantitative Imaging in Medicine and Surgery. 11 (3), 1078-1101 (2021).
  20. Yamakoshi, H., et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. Journal of the American Chemical Society. 133 (16), 6102-6105 (2011).
  21. Shi, L., et al. Optical imaging of metabolic dynamics in animals. Nature Communications. 9 (1), 2995 (2018).
  22. Bagheri, P., Hoang, K., Fung, A. A., Hussain, S., Shi, L. Visualizing cancer cell metabolic dynamics regulated with aromatic amino acids using DO-SRS and 2PEF microscopy. Frontiers in Molecular Biosciences. 8, 779702 (2021).
  23. Li, Y., et al. Direct imaging of lipid metabolic changes in drosophila ovary during aging using DO-SRS microscopy. Frontiers in Aging. 2, 819903 (2022).
  24. Li, Y., Zhang, W., Fung, A. A., Shi, L. DO-SRS imaging of metabolic dynamics in aging Drosophila. Analyst. 146 (24), 7510-7519 (2021).
  25. Zhang, L., et al. Spectral tracing of deuterium for imaging glucose metabolism. Nature Biomedical Engineering. 3 (5), 402-413 (2019).
  26. Fung, A. A., et al. Imaging sub-cellular methionine and insulin interplay in triple negative breast cancer lipid droplet metabolism. Frontiers in Oncology. 12, 858017 (2022).
  27. Li, Y., Zhang, W., Fung, A. A., Shi, L. DO-SRS imaging of diet regulated metabolic activities in Drosophila during aging processes. Aging Cell. 21 (4), e13586 (2022).
  28. Shi, L., Wei, M., Min, W. Highly-multiplexed tissue imaging with raman dyes. Journal of Visualized Experiments. (182), e63547 (2022).
  29. Rysman, E., et al. De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation. Cancer Research. 70 (20), 8117-8126 (2010).
  30. Lisec, J., Jaeger, C., Rashid, R., Munir, R., Zaidi, N. Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia. BMC Cancer. 19 (1), 501 (2019).
  31. Thiam, A. R., Dugail, I. Lipid droplet-membrane contact sites - from protein binding to function. Journal of Cell Science. 132 (12), (2019).
  32. Schott, M. B., et al. Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes. The Journal of Cell Biology. 218 (10), 3320-3335 (2019).
  33. Hoang, K., et al. Subcellular resolution DO-SRS and 2PEF imaging of metabolic dynamics regulated by L-methionine in amyotrophic lateral sclerosis. Optical Biopsy XXI: Toward Real-Time Spectroscopic Imaging and Diagnosis. SPIE. 1237303, 6-13 (2023).
  34. Jang, H., et al. Super-resolution stimulated Raman scattering microscopy with A-PoD. bioRxiv. , (2022).
  35. Li, Y., et al. Optical metabolic imaging uncovers sex- and diet-dependent lipid changes in aging drosophila brain. bioRxiv. , (2022).
  36. Zhang, W., et al. Multi-molecular hyperspectral PRM-SRS imaging. bioRxiv. , (2022).
  37. Wei, M., et al. Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy. Proceedings of the National Academy of Sciences. 116 (14), 6608-6617 (2019).
  38. Chang, T., et al. Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy. Biomaterials. 34 (34), 8607-8616 (2013).
  39. Leica TCS SP8 CARS CARS Microscope - Label Free Imaging. Leica Microsystems Available from: https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-cars/downloads/ (2023)

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved