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Introduction
Protocol
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Bioengineering

In vivo Billeddannelse af biologisk væv med kombineret to-fotonfluorescens og stimuleret Raman-spredningsmikroskopi

Published: December 20, 2021
doi:
10.3791/63411
, , ,
1Department of Electronic and Computer Engineering,The Hong Kong University of Science and Technology, 2State Key Laboratory of Molecular Neuroscience,The Hong Kong University of Science and Technology, 3Center of Systems Biology and Human Health,The Hong Kong University of Science and Technology, 4Molecular Neuroscience Center,The Hong Kong University of Science and Technology, 5Department of Biology, School of Life Sciences,Southern University of Science and Technology

Summary

Stimuleret Raman scattering (SRS) mikroskopi tillader etiketfri billeddannelse af biomolekyler baseret på deres iboende vibration af specifikke kemiske bindinger. I denne protokol beskrives den instrumentelle opsætning af et integreret SRS og to-fotonfluorescensmikroskop for at visualisere cellulære strukturer i rygmarven hos levende mus.

Abstract

Stimuleret Raman scattering (SRS) mikroskopi muliggør etiketfri billeddannelse af det biologiske væv i dets naturlige mikromiljø baseret på iboende molekylær vibration, hvilket giver et perfekt værktøj til in vivo-undersøgelse af biologiske processer ved subcellulær opløsning. Ved at integrere to-foton ophidset fluorescens (TPEF) billeddannelse i SRS-mikroskopet kan den dobbeltmodale in vivo-billeddannelse af væv erhverve kritisk biokemisk og biofysisk information fra flere perspektiver, som hjælper med at forstå de dynamiske processer, der er involveret i cellulær metabolisme, immunrespons og vævsombygning osv. I denne videoprotokol introduceres opsætningen af et TPEF-SRS-mikroskopsystem samt in vivo-billeddannelsesmetoden for dyrets rygmarv. Rygmarven, som en del af centralnervesystemet, spiller en kritisk rolle i kommunikationen mellem hjernen og det perifere nervesystem. Myelinskede, der er rigelig i phospholipider, omgiver og isolerer axonen for at tillade saltholdig ledning af aktionspotentialer. In vivo-billeddannelse af myelinskeder i rygmarven er vigtig for at studere progressionen af neurodegenerative sygdomme og rygmarvsskade. Protokollen beskriver også dyrepræparationer og in vivo TPEF-SRS-billeddannelsesmetoder til at erhverve biologiske billeder i høj opløsning.

Introduction

Ramanmikroskopi1,2 fremstår som en kraftfuld etiketfri metode til at afbilde biologisk væv baseret på de karakteristiske frekvenser af forskellige kemiske bindinger i biomolekyler. På grund af sin ikke-invasive og veladaptive billeddannelsesevne er Raman-mikroskopi i vid udstrækning blevet anvendt til billeddannelse af lipidberigede komponenter i biologiske væv som myelinskede3,4,5, adipocytter6,7 og lipiddråber8,9,10 . Stimuleret Raman scattering (SRS) signal erhvervet som stimuleret Raman gain (SRG) eller stimuleret Raman tab (SRL) er baggrundsfrit, hvilket viser perfekt spektral lighed med spontan Raman spredning11,12. Derudover er SRL og SRG lineært afhængige af analytkoncentrationen, hvilket muliggør kvantitativ analyse af biokemiske komponenter9,11,13. To-foton ophidset fluorescensmikroskopi (TPEF) er blevet anvendt i vid udstrækning til in vivo biologisk billeddannelse på grund af dets iboende optiske sektioneringsevne, dybe penetrationsdybde og lave fototoksicitet14,15,16. Udførelsen af TPEF-billeddannelse afhænger imidlertid af egenskaberne ved fluorescerende tags, og antallet af opløselige farver er begrænset på grund af bredbåndsfluorescensspektrene8,17,18,19. Etiketfri SRS-billeddannelse og fluorescensbaseret TPEF-billeddannelse er to komplementære billeddannelsesmetoder, og deres kombination kan give rigelig biofysisk og biokemisk information om væv. Disse to billeddannelsesmetoder er begge baseret på de ikke-lineære optiske (NLO) processer, som muliggør enkel integration i et mikroskopsystem. Kombinationen af SRS- og TPEF-billeddannelsen, den såkaldte dual-modal imaging, muliggør højdimensionel billeddannelse og profilering af celler og væv, hvilket letter en omfattende forståelse af komplekse biologiske systemer. Specifikt kan picosekund (ps) SRS-mikroskopi opnå kemisk bindingsbilleddannelse med høj spektral opløsning sammenlignet med femtosekund (fs) SRS-teknik11, hvilket gør det muligt at differentiere flere biokemiske komponenter i biologisk væv, især i det overfyldte fingeraftryksområde20,21. Sammenlignet med et andet almindeligt anvendt dobbeltmodalt NLO-mikroskopsystem med integration af sammenhængende anti-Stokes scattering (CARS) mikroskop viser SRS desuden overlegen ydeevne i forhold til CARS med hensyn til spektral- og billedfortolkning samt detektionsfølsomhed11. SRS-TPEF-mikroskopet er blevet brugt som et kraftfuldt værktøj til at studere forskellige biologiske systemer, såsom Caenorhabditis elegans9,22, Xenopus laevis tadpole brain5, musehjerne23,24, rygmarv25,26, perifer nerve27 og fedtvæv7 osv.

Rygmarven udgør sammen med hjernen centralnervesystemet (CNS). Visualisering af cellulære aktiviteter i CNS in vivo under fysiologiske og patologiske tilstande er afgørende for at forstå mekanismerne for CNS-lidelser28,29,30 og for at udvikle tilsvarende terapier31,32,33. Myelinskede, der ombryder og isolerer axoner til højhastighedsvirkende potentiel ledning, spiller en væsentlig rolle i udviklingen af CNS. Demyelination betragtes som et kendetegn ved hvide substansforstyrrelser, såsom multipel sklerose34. Efter rygmarvsskade35 kan myelinaffald desuden modulere makrofagaktivering, hvilket bidrager til kronisk inflammation og sekundær skade36. Derfor er in vivo-billeddannelse af myelinskede sammen med neuroner og gliaceller i levende musemodeller til stor hjælp til at forstå de dynamiske processer i CNS-lidelser.

I denne protokol beskrives de grundlæggende opsætningsprocedurer for et hjemmebygget TPEF-SRS-mikroskop, og de dobbeltmodale in vivo-billeddannelsesmetoder til musens rygmarv introduceres.

Protocol

Alle dyreforsøg, der udføres i dette arbejde, udføres i overensstemmelse med retningslinjerne fra Laboratory Animal Facility ved Hong Kong University of Science and Technology (HKUST) og er godkendt af HKUST’s dyreetiske komité. Sikkerhedstræning i laserhåndtering er nødvendig for at opsætte og betjene TPEF-SRS-mikroskopet. Brug altid lasersikkerhedsbriller med passende bølgelængdeområde, når du beskæftiger dig med laser. 1. Opsætning af TPEF-SRS-mikroskopet (for opsætning…

Representative Results

In vivo dual-modal billeddannelse af spinal axoner såvel som myelinskeder udføres ved hjælp af Thy1-YFPH transgene mus, som udtrykker EYFP i dorsale rodganglion afferente neuroner (figur 3). Disse mærkede afferente neuroner videresender den sensoriske information fra den perifere nerve til rygmarven, med den centrale gren placeret i rygmarven dorsal søjle. Med TPEF-SRS-mikroskopet kan tæt distribueret myelinskede tydeligt visualiseres ved hjælp af etiketfri SRS-billeddannelse…

Discussion

I denne protokol beskrives den grundlæggende opsætning af TPEF-SRS-mikroskopet detaljeret. Til SRS-billeddannelse overlappes pumpen og Stokes-strålerne tidsmæssigt og rumligt inde i OPO’en. Denne overlapning kan dog forstyrres efter at have passeret mikroskopsystemet. Derfor er både rumlig og tidsmæssig optimering af colokaliseringen af pumpen og Stokes-bjælkerne nødvendig og kritisk for at opnå optimal SRS-billeddannelse. Den tidsmæssige forsinkelse mellem pumpen og Stokes-strålen er relateret til den optisk…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af Hong Kong Research Grants Council gennem tilskud 16103215, 16148816, 16102518, 16102920, T13-607/12R, T13-706/11-1, T13-605/18W, C6002-17GF, C6001-19E, N_HKUST603/19, Innovation and Technology Commission (ITCPD/17-9), Area of Excellence Scheme fra University Grants Committee (AoE/M-604/16, AOE/M-09/12) og Hong Kong University of Science & Technology (HKUST) gennem tilskud RPC10EG33.

Materials

#2 Forceps Dumont 11223-20 For laminectomy
10X objective Nikon CFI Plan Apo Lambda 10X
25X objective Olympus XLPLN25XSVMP2
Burn cream Betadine
Camera Sony α6300
Current amplifier Stanford research SR570
Current photomultiplier modules Hamamatsu H11461-01
D2 665 nm long-pass dichroic mirror Semrock FF665-Di02-25×36 For directing epi-fluorescence signal to the detection module
D3 700 nm short-pass dichroic mirror Edmund 69-206 For separating SRS from TPEF detection path
Depilating cream Veet
FS1 975 nm short-pass filter Edmund 86-108 For blocking stokes beam
FS1 Bandpass filter Semrock FF01-850/310 For blocking stokes beam
Fs2 Bandpass filter Semrock FF01-525/50 For selecting YFP signal
Fs2 Shortpass filter Semrock FF01-715/SP-25 For blocking fs excitation laser beam
Half-wave plate Thorlabs SAHWP05M-1700
High-speed photodetector MenloSystems FPD 310-F For checking Stokes beam modulation
Iodine Betadine
IR Scope FJW FIND-R-SCOPE Infrared Viewer 2X Kit Model 84499C2X
Iris Thorlabs CPA1
L1 Thorlabs AC254-060-B-ML
L10 Thorlabs LA4052-A
L2 Thorlabs LA1422-B
L3 Thorlabs AC254-050-B
L4 Thorlabs AC254-060-B-ML
L7 f=100 mm, AB coating
L8 Thorlabs LA4874-A
L9 Thorlabs AC254-035-B-ML
Lock-in amplifier APE
Mirror Thorlabs PF10-03-P01
Motorized flipper Thorlabs MFF101/M
multifunctional acquisition card National Instrument PCIe-6363
Oscilloscope Tektronix TDS2012C
Photodiode APE For detecting SRS signal
Picosecond laser source APE picoEmerald
Polarizing beam splitter Thorlabs CCM1-PBS252/M
Power meter Newport 843-R
Saline Braun
Scan lens L5 Thorlabs SL50-CLS2
Scanning mirror Cambridge Technology 6215H
Silicone gel World Precision Inc. KWIK-SIL
Ti:sapphire fs laser Coherent Chameleon Ultra II
Tube lens L6 Thorlabs TTL200-S8
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Tags

In Vivo ImagingTwo-photon FluorescenceStimulated Raman ScatteringMicroscopySubcellular ResolutionChemical SpecificityCellular MetabolismImmune ResponseNeurodegenerative DiseasesSpinal Cord InjuryTitanium Sapphire LaserOPOPhotodetectorOscilloscopeEOMPump LaserWavelengthOptical Alignment
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Wu, W., Li, X., Qu, J. Y., He, S. In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy. J. Vis. Exp. (178), e63411, doi:10.3791/63411 (2021).
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