Meget multiplexeret vævsbilleddannelse med Raman-farvestoffer
Summary
Elektronisk præresonansstimuleret Raman scattering (epr-SRS) billeddannelse af regnbuelignende Raman farvestoffer er en ny platform for stærkt multiplexeret epitopbaseret proteinbilleddannelse. Her præsenterer vi en praktisk vejledning, herunder antistofforberedelse, vævsprøvefarvning, SRS-mikroskopsamling og epr-SRS-vævsbilleddannelse.
Abstract
Visualisering af et stort omfang af specifikke biomarkører i væv spiller en afgørende rolle i udforskningen af de indviklede organisationer af komplekse biologiske systemer. Derfor er stærkt multiplexede billeddannelsesteknologier i stigende grad blevet værdsat. Her beskriver vi en ny platform for stærkt multiplexeret vibrationsbilleddannelse af specifikke proteiner med sammenlignelig følsomhed over for standard immunofluorescens via elektronisk præresonansstimuleret Raman scattering (epr-SRS) billeddannelse af regnbuelignende Raman farvestoffer. Denne metode omgår grænsen for spektraltopløselige kanaler i konventionel immunfluorescens og giver en one-shot optisk tilgang til at forhøre flere markører i væv med subcellulær opløsning. Det er generelt kompatibelt med standardvævspræparater, herunder paraformaldehyd-fast væv, frosne væv og formalin-fast paraffinindlejret (FFPE) humant væv. Vi forestiller os, at denne platform vil give et mere omfattende billede af proteininteraktioner mellem biologiske prøver, især for tykt intakt væv. Denne protokol giver arbejdsgangen fra antistofforberedelse til vævsprøvefarvning, til SRS-mikroskopsamling til epr-SRS-vævsbilleddannelse.
Introduction
Komplekse vævssystemer består af forskellige cellulære subpopulationer, hvis rumlige placeringer og interaktionsnetværk er dybt sammenflettet med deres funktioner og dysfunktioner 1,2. For at afsløre vævsarkitekturen og forhøre dens kompleksitet er viden om de rumlige placeringer af proteiner ved enkeltcelleopløsning afgørende. Derfor er stærkt multiplexerede proteinbilleddannelsesteknologier i stigende grad blevet værdsat og kan blive en hjørnesten for at studere vævsbiologi 3,4,5. Nuværende almindelige multiplexerede proteinbilleddannelsesmetoder kan klassificeres i to hovedkategorier. Den ene er seriel immunofluorescensbilleddannelse, der er afhængig af flere runder vævsfarvning og billeddannelse, og den anden er billeddannelse af massecytometri kombineret med tungmetalmærkede antistoffer 6,7,8,9,10,11,12.
Her introduceres en alternativ strategi for multiplexeret antistofbaseret proteinbilleddannelse. I modsætning til den fremherskende fluorescensbilleddannelsesmodalitet, som kun kan visualisere 4-5 kanaler samtidigt på grund af de brede excitations- og emissionsspektre (fuld bredde ved halv maksimum (FWHM) ~ 500 cm-1), udviser Raman-mikroskopi meget smallere spektral linjebredde (FWHM ~ 10 cm-1) og giver dermed skalerbar multiplexitet. For nylig, ved at udnytte det smalle spektrum, er der udviklet en ny ordning med Raman-mikroskopi ved navn elektronisk præresonansstimuleret Raman-spredning (epr-SRS) mikroskopi, hvilket giver en stærk strategi for multiplexeret billeddannelse13. Ved at undersøge de elektronisk koblede vibrationstilstande for Raman-farvestoffer opnår epr-SRS en drastisk forbedringseffekt på 1013 gange på Raman-tværsnit og overvinder følsomhedsflaskehalsen i konventionel Raman-mikroskopi (figur 1A) 13,14,15. Som følge heraf er detektionsgrænsen for epr-SRS blevet skubbet til sub-μM, hvilket muliggør Raman-detektion af interessante molekylære markører såsom specifikke proteiner og organeller inde i celler13,16. Ved anvendelse af Raman-farvestofkonjugerede antistoffer blev epr-SRS-billeddannelse af specifikke proteiner i celler og væv (kaldet immuno-eprSRS) især demonstreret med sammenlignelig følsomhed over for standard immunofluorescens (figur 1B) 13,17. Ved at indstille pumpens bølgelængde med kun 2 nm vil epr-SRS-signalet være helt slukket (figur 1B), som viser høj vibrationskontrast.
På sondesiden er der udviklet et sæt regnbuelignende Raman-sonder kaldet Manhattan Raman scattering (MARS) farvestoffer til antistofkonjugering 13,18,19,20. Denne unikke Raman-palet består af nye farvestoffer med π-konjugerede tredobbelte bindinger (supplerende materiale), der hver viser en enkelt og smal epr-SRS-top i det bioortogonale Raman-spektralområde (figur 1C). Ved at ændre strukturen af kernekromoforen og isotopisk redigere begge atomer af den tredobbelte binding (supplerende materiale) er der udviklet spektralt adskilte Raman-sonder. Ved at udnytte den skalerbare multiplexitet tilbyder epr-SRS-mikroskopi kombineret med MARS-farvepaletten en optisk strategi til one-shot multiplex-proteinbilleddannelse i celler og væv.
Immuno-eprSRS giver en alternativ strategi til de nuværende multiplex proteinbilleddannelsesmetoder med unikke styrker. Sammenlignet med fluorescenstilgange med cyklisk farvning, billeddannelse og signalfjernelse sikrer denne Raman-baserede platform enkeltrunde farvning og billeddannelse. Derfor omgår det den praktiske kompleksitet i cykliske procedurer og forenkler i vid udstrækning protokollen og åbner dermed nye territorier for multiplexeret proteinbilleddannelse. Ved at udnytte en Raman-farvestof-skræddersyet vævsclearingsprotokol er immuno-eprSRS f.eks. blevet udvidet til tre dimensioner til stærkt multiplexeret proteinkortlægning i tykt intakt væv17. Over 10 proteinmål blev visualiseret langs millimetertykt musehjernevæv17. For nylig er kobling af immuno-eprSRS med en optimeret biomolekyle-retentionsekspansionsmikroskopi (ExM) protokol21, one-shot nanoskala billeddannelse af flere mål også blevet demonstreret22. Sammenlignet med billeddannelse af massespektroskopi 4,9 er epr-SRS ikke-destruktiv og har iboende optisk sektionsevne. Desuden er epr-SRS mere tidseffektiv på vævsscanning. Typisk tager et vævsområde på 0,25 mm2 med en pixelstørrelse på 0,5 μm kun et par minutter at afbilde for en enkelt epr-SRS-kanal. For eksempel er den samlede billedbehandlingstid for fire SRS-kanaler plus fire fluorescenskanaler i figur 4 ca. 10 minutter.
Protocol
Representative Results
Discussion
Her præsenterer vi immuno-eprSRS-protokollen, som er bredt anvendelig på almindelige vævstyper, herunder friskbevaret musevæv, FFPE humant væv og frosne musevæv. Immuno-eprSRS er blevet valideret for et panel af epitoper i celler og væv, som anført i tabel 1. Denne one-shot platform er især velegnet til applikationer, hvor cykliske strategier ikke fungerer godt. For eksempel kræver cyklisk fluorescens tykt væv, da flere runder af 3D-immunmærkning er upraktiske lange17….
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi takker Ruth A. Singer og Richard K.P. Benninger for at have leveret musevæv i bugspytkirtlen. W.M. anerkender støtte fra NIH R01 (GM128214), R01 (GM132860), R01 (EB029523) og US Army (W911NF-19-1-0214).
Materials
| 16% Paraformaldehyde, EM Grade | Electron Microscopy Sciences | 15710 | |
| α-tubulin | Abcam | ab18251 | Primary antibodies |
| α-tubulin | BioLegend | 625902 | Primary antibodies |
| β-III-tubulin | BioLegend | 657402 | Primary antibodies |
| β-III-tubulin | Abcam | ab41489 | Primary antibodies |
| β-tubulin | Abcam | ab131205 | Primary antibodies |
| Agarose, low gellling temperature | Sigma Aldrich | A9414 | For brain embedding |
| Anti-a-tubulin antibody produced in rabbit (α-tubulin) | Abcam | ab52866 | Primary antibodies |
| Anti-Calbindin antibody produced in mouse (Calbindin) | Abcam | ab82812 | Primary antibodies |
| Anti-GABA B receptor R2 antibody produced in guinea pig (GABA B receptor R2) | Millipore Sigma | AB2255 | Primary antibodies |
| Anti-GFAP antibody produced in goat (GFAP) | Thermo Scientific | PA5-18598 | Primary antibodies |
| Anti-Glucagon antibody produced in mouse (Glucagon) | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
| Anti-insulin antibody produced in guinea pig (insulin) | DAKO | IR00261-2 | Primary antibodies |
| Anti-MBP antibody produced in rat (MBP) | Abcam | ab7349 | Primary antibodies |
| Anti-NeuN antibody produced in rabbit (NeuN) | Thermo Scientific | PA5-78639 | Primary antibodies |
| Anti-Pancreatic polypeptide (PP) antibody produced in goat- Pancreatic polypeptide (PP) | Sigma Aldrich | SAB2500747 | Primary antibodies |
| Anti-Pdx1 antibody produced in rabbit (Pdx1) | Milipore | 06-1379 | Primary antibodies |
| Anti-Somatostatin antibody produced in rat (Somatostatin) | Abcam | ab30788 | Primary antibodies |
| Anti-Vimentin antibody produced in chicken (Vimentin) | Abcam | ab24525 | Primary antibodies |
| 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 |
| C57BL/6J mice | Jackson Laboratory | 000664 | |
| Centrifuge | |||
| Condenser | Olympus | oil immersion, 1.4 N.A. | |
| Cytokeratin 18 | Abcam | ab7797 | Primary antibodies |
| Cytokeratin 18 | Abcam | ab24561 | Primary antibodies |
| DC power supply | TopWard | 6302D | Bias voltage is 64 V |
| Dichroic mount | Thorlabs | KM100CL | Kinematic Mount for up to 1.3" (33 mm) Tall Rectangular Optics, Left Handed |
| Donkey anti-Chicken IgY (H+L) | Jackson ImmunoResearch | 703-005-155 | Secondary antibodies for MARS conjugation |
| Donkey anti-Goat IgG (H+L) | Jackson ImmunoResearch | 705-005-147 | Secondary antibodies for MARS conjugation |
| Donkey anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch | 706-005-148 | Secondary antibodies for MARS conjugation |
| Donkey anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-005-151 | Secondary antibodies for MARS conjugation |
| Donkey anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-005-152 | Secondary antibodies for MARS conjugation |
| Donkey anti-Rat IgG (H+L) | Jackson ImmunoResearch | 712-005-153 | Secondary antibodies for MARS conjugation |
| Donkey anti-Sheep IgG (H+L) | Jackson ImmunoResearch | 713-005-147 | Secondary antibodies for MARS conjugation |
| DPBS | Fisher Scientific | 14-190-250 | |
| EpCAM | Abcam | ab71916 | Primary antibodies |
| Ethanol | Sigma Aldrich | 443611 | |
| Fast-speed look-in amplifier | Zurich Instruments | HF2LI | DC – 50 MHz |
| FFPE Kidney Sample | USBiomax | HuFPT072 | |
| Fibrillarin | Abcam | ab5821 | Primary antibodies |
| Giantin | Abcam | ab24586 | Primary antibodies |
| Glucagon | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
| H2B | Abcam | ab1790 | Primary antibodies |
| HeLa | ATCC | ATCC CCL-2 | |
| High O.D. bandpass filter | Chroma Technology | ET890/220m | Filter the Stokes beam and transmit the pump beam |
| Hydrophobic pen | Fisher Scientific | NC1384846 | |
| Insulin | ThermoFisher | 701265 | Primary antibodies |
| Integrated SRS laser system | Applied Physics & Electronics, Inc. | picoEMERALD | picoEMERALD provides an output pulse train at 1,064 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 1,064-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 pulse trains is 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 | |
| Kinematic mirror mount | Thorlabs | POLARIS-K1-2AH | 2 Low-Profile Hex Adjusters |
| Lectin from Triticum vulgaris (wheat) | Sigma Aldrich | L0636-5 mg | |
| Long-pass dichroic beam splitter | Semrock | Di02-R980-25×36 | 980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter |
| MAP2 | BioLegend | 801810 | Primary antibodies |
| Microscopy imaging software | Olympus | FluoView | |
| NanoQuant Plate | Tecan | For absorbance-based, small volume analyses in a plate reader. | |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | |
| NucBlue Fixed Cell ReadyProbes Reagent (DAPI) | Thermo Scientific | R37606 | |
| Nunc 4-Well Dishes | Fisher Scientific | 12-566-300 | |
| Objective lens | Olympus | XLPlan N | x25, 1.05-NA, MP, working distance = 2 mm |
| Paint brush | |||
| Periscope assembly | Thorlabs | RS99 | includes the top and bottom units, Ø1" post, and clamping fork. |
| pH meter | |||
| Plate reader | Tecan | Infinite 200 PRO | An easy-to-use multimode plate reader. Absorbance measurement capabilities over a spectral range of 230–1000 nm. |
| ProLong Gold antifade reagent | Thermo Scientific | P36930 | |
| PSD95 | Invitrogen | 51-6900 | Primary antibodies |
| Sephadex G-25 Medium | GE Life Sciences | 17-0033-01 | gel filtration resin for desalting and buffer exchange |
| Shielded box with BNC connectors | Pomona Electronics | 2902 | Aluminum Box With Cover, BNC Female/Female |
| Si photodiode | Thorlabs | FDS1010 | 350–1100 nm, 10 mm x 10 mm Active Area |
| Synapsin 2 | ThermoFisher | OSS00073G | Primary antibodies |
| Tissue Path Superfrost Plus Gold Slides | Fisher Scientific | 22-035813 | Adhesive slide to attract and chemically bond fresh or formalin-fixed tissue sections firmly to the slide surface (tiisue bindling glass slides) |
| Triton X-100 | Fisher Scientific | BP151-500 | |
| Vibratome | Leica | VT1000 | |
| Vimentin | Abcam | ab8069 | Primary antibodies |
| Xylenes | Sigma Aldrich | 214736 |
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