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Summary
Abstract
Introduction
Protocol
Results
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Acknowledgements
Materials
References
자동 번역
생물학

Direkte stokastisk optisk rekonstruktion Mikroskopi af ekstracellulære vesikler i tre dimensioner

Published: August 26, 2021
doi:
10.3791/62845
, ,
1Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center,The University of North Carolina at Chapel Hill School of Medicine

Summary

Direkte stokastisk optisk rekonstruktionsmikroskopi (dSTORM) bruges til at omgå den typiske diffraktionsgrænse for lysmikroskopi og til at se exosomer på nanometerskalaen. Det kan anvendes i både to og tre dimensioner til at karakterisere exosomer.

Abstract

Ekstracellulære vesikler (elbiler) frigives af alle celletyper og spiller en vigtig rolle i cellesignalering og homøostase. Visualiseringen af elbiler kræver ofte indirekte metoder på grund af deres lille diameter (40-250 nm), hvilket er under diffraktionsgrænsen for typisk lysmikroskopi. Vi har udviklet en mikroskopibaseret visualisering af elbiler med superopløsning for at omgå diffraktionsgrænsen i både to og tre dimensioner. Ved hjælp af denne tilgang kan vi løse den tredimensionelle form af elbiler til inden for +/- 20 nm opløsning på XY-aksen og +/- 50 nm opløsning langs Z-aksen. Afslutningsvis foreslår vi, at mikroskopi med superopløsning betragtes som en karakteriseringsmetode for elbiler, herunder exosomer, samt indhyllede vira.

Introduction

Ekstracellulære vesikler (elbiler) er membranbundne vesikler frigivet af alle celletyper. De indeholder lipider, proteiner, metabolitter og nukleinsyrer og overfører disse materialer lokalt mellem celler og distally mellem væv og organer. Der findes tre primære undertyper af elbiler: apoptotiske kroppe, mikrovesicles og exosomer1,2. Her fokuserer vi vores diskussion på exosomer og deres tilhørende proteiner.

Exosomer er udskilles vesikler stammer fra indad spirende af tidlige endosomes i multivesicular body (MVB). MVB smelter derefter sammen med plasmamembranen og frigiver exosomer i det ekstracellulære rum for at rejse til andre celler3,4. Exosomer findes på et spektrum af størrelser fra 40 til 150 nm og er beriget med endosomal transmembrane proteiner kendt som tetraspaniner (CD9, CD63, CD81), membran-bundet endosomal sortering kompleks kræves til transport (ESCRT), og lipid flåde-associerede proteiner1,2,5,6,7.

Karakterisering af exosomers biokemiske sammensætning er blevet et populært område for forskere til bedre at forstå deres funktionelle natur. Mange metoder findes til visualisering og karakterisering af exosomer, herunder nanoskala flowcytometri, nanopartikler tracking analyse (NTA), scanning og transmission elektron mikroskopi (TEM), overflade plasmon resonans, resistiv puls sensing, og traditionelle lysmikroskopi, som hver indeholder iboende fordele og ulemper8,9. TEM og cryo-EM kan opnå nanometerbaseret opløsning, men kræver ofte dehydrerende og frysefrakturtrin og derved krympe eller lysing af elbiler10,11. NTA er afhængig af lysspredning, der giver mulighed for karakterisering af hundredvis af elbiler ad gangen, men er en indirekte måling af partikelstørrelse og kan ikke let skelne mellem elbiler, vira og proteinaggregater12,13,14,15,16. Nanoskala flowcytometri anvender lysspredning fra en excitationssti, som derefter kan oversættes til størrelsesmålinger, men er en ny teknologi, og der er ringe enighed om, hvilken størrelse partikler der er inden for det lineære område af detektion for forskellige instrumenter12,17,18.

Traditionel lysmikroskopi ved hjælp af fluorescerende proteiner eller farvestoffer har været en af de mest anvendte teknikker til visualisering af subcellulære rum, proteinkomplekser og signalmaskineri i en celle. Mens denne teknik viser sig nyttig til at visualisere lokalisering af komplekser, diffraktion grænse for traditionelle lysmikroskopi (ca. 250-400 nm) forhindrer klar opløsning af proteiner eller strukturer i den typiske størrelse rækkevidde af en exosome (40-150 nm)12,19,20.

Super-opløsning mikroskopi, nemlig direkte stokastisk optisk rekonstruktion mikroskopi (dSTORM), adskiller sig fra konventionelle lysmikroskopi ved at anvende photoswitchable egenskaber specifikke fluorophores og opdage disse blinkende begivenheder til at rekonstruere billeder ned til nanometer præcision21. Photoswitching begivenheder indsamles ved hjælp af en høj-framerate detektion kamera i løbet af titusinder af individuelle eksponeringer, og et punkt spredning funktion bruges til at kortlægge med høj tillid den nøjagtige placering af photoswitching fluorophore19,20,22. Dette gør det muligt for dSTORM at omgå diffraktionsgrænsen for lysmikroskopi. Flere grupper har rapporteret brugen af superopløsningsteknikker til visualisering og sporing af exosomer og deres tilknyttede proteiner22,23,24,25. Den endelige opløsning afhænger af fluorophorens biofysiske egenskaber, men varierer ofte fra +/-10-100 nm langs XY-aksen, hvilket giver mulighed for opløsning med enkeltmolekyler.

Evnen til at løse individuelle fluorophorer på denne skala på XY-aksen har revolutioneret mikroskopi. Der er dog kun få data om den tredimensionelle (3D) dSTORM af en exosome. Derfor forsøgte vi at etablere en standardprocedure (SOP) for dSTORM-baseret visualisering og karakterisering af rensede elbiler, herunder exosomer til nanometerpræcision i 3D.

Protocol

1 Formering og vedligeholdelse af cellelinjer Erhverve human osteosarcoma celler (U2OS) og placere cellerne i vækstmediet suppleret med 10% exosome-fri Foster kvæg Serum og 1x Penicillin / Streptomycin opløsning.BEMÆRK: Exosome-fri Fosterkvæg Serum blev genereret efter protokollen præsenteret i McNamara et. al.26. U2OS-cellerne vedligeholdes i en kobberbelagt inkubator ved 37 °C i 5 % CO2 og passageceller i T175-kolber26,…

Representative Results

Målet med denne undersøgelse var at evaluere effektiviteten af super-opløsning mikroskopi i visualisering af individuelle elbiler med nanometer opløsning i tre dimensioner (3-D). For at analysere formen og størrelsen af de enkelte elbiler, vi ansat photoswitchable farvestof og inkuberet elbiler med en langt rød, membran intercalating farvestof, og fjernet overskydende farvestof gennem kromatografi29. Den affinitet-fanget anti-CD81 og rød-farvede elbiler blev derefter set i super-opløsning …

Discussion

Elbiler er blevet et populært studieområde på grund af deres vigtige rolle i mange intracellulære processer og celle-til-celle signalering1,30. Men deres visualisering viser sig at være vanskeligt, da deres lille størrelse falder under diffraktionsgrænsen for lysmikroskopi. Direkte stokastisk optisk rekonstruktion mikroskopi (dSTORM) er en direkte metode til visualisering, der omgår diffraktion grænse ved at fange photoswitching begivenheder af individue…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi vil gerne takke Oxford Nanoimaging for deres konstruktive feedback og vejledning. Dette arbejde blev finansieret af 5UM1CA121947-10 til R.P.M. og 1R01DA040394 til D.P.D.

Materials

15 µ-Slide 8 well plates Ibidi 80827
1X PBS Gibco 14190-144
1X Penicillin Streptomycin solution Gibco 15140-122
50 mL conical tube Thermo Fisher 339652
500 mL 0.22 µm vacuum filtration apparatus Genesee 25-227
750 kDa hollow-fiber cartridge cutoff filter Cytiva 29-0142-95
AKTA Flux S Cytiva 29-0384-37
AKTA Start Cytiva 29022094-ECOMINSSW
Anti-CD81 magnetic beads Thermo Fisher 10616D
B-cubed buffer ONI  BCA0017
CellMask Red Thermo Fisher C10046
Dubelco's Modified Eagle Medium Thermo Fisher 10566016
Fetal Bovine Serum VWR 97068-085
Frac 30 Fraction collector Cytiva 29022094-ECOMINSSW
Glycine pH=2.0 Thermo Fisher BP381-5
HiTrap CaptoCore 700 Column Cytiva 17548151
Molecular Biology Grade Water Corning 9820003
Nanoimager Oxford Nanoimaging Custom
Paraformaldehhyde Electron Microscopy Sciences 15710
Polyethylene glycol Thermo Fisher BP233-1
RNase A Promega A797C
T175 Flasks Genesee 25-211
Tetraspek microspheres Invitrogen T7279
Tris- HCl pH=7.5 Thermo Fisher BP153-1
Unicorn V Cytiva 29022094-ECOMINSSW
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Tags

Extracellular VesiclesDSTORMSuper-resolution MicroscopyThree-dimensional VisualizationVirus ParticlesDisease BiomarkersAffinity PurificationParaformaldehyde FixationImaging BufferCalibration Beads
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Chambers, M. G., McNamara, R. P., Dittmer, D. P. Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions. J. Vis. Exp. (174), e62845, doi:10.3791/62845 (2021).
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