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Bioengineering

Biomolekylær billeddannelse af cellulær optagelse af nanopartikler ved hjælp af multimodal ikke-lineær optisk mikroskopi

Published: May 16th, 2022

DOI:

10.3791/63637

1Biomedical Physics, School of Physics and Astronomy, University of Exeter
* These authors contributed equally

Denne artikel præsenterer integrationen af et spektralfokuseringsmodul og en dual-output pulslaser, der muliggør hurtig hyperspektral billeddannelse af guldnanopartikler og kræftceller. Dette arbejde har til formål at demonstrere detaljerne i multimodale ikke-lineære optiske teknikker på et standard laserscanningsmikroskop.

Sondering af guldnanopartikler (AuNP'er) i levende systemer er afgørende for at afsløre samspillet mellem AuNP'er og biologiske væv. Ved at integrere ikke-lineære optiske signaler såsom stimuleret Raman-spredning (SRS), to-foton-exciteret fluorescens (TPEF) og forbigående absorption (TA) i en billeddannelsesplatform kan den desuden bruges til at afsløre biomolekylær kontrast mellem cellulære strukturer og AuNP'er på en multimodal måde. Denne artikel præsenterer en multimodal ikke-lineær optisk mikroskopi og anvender den til at udføre kemisk specifik billeddannelse af AuNP'er i kræftceller. Denne billeddannelsesplatform giver en ny tilgang til udvikling af mere effektive funktionaliserede AuNP'er og bestemmelse af, om de er inden for vaskulaturer omkring tumor-, pericellulære eller cellulære rum.

Guldnanopartikler (AuNP'er) har vist stort potentiale som biokompatible billeddannende sonder, for eksempel som effektive overfladeforstærkede Raman-spektroskopi (SERS) substrater i forskellige biomedicinske applikationer. Større anvendelser omfatter områder som biosensing, bioimaging, overfladeforbedrede spektroskopier og fototermisk terapi til kræftbehandling1. Desuden er sondering af AuNP'er i levende systemer afgørende for at vurdere og forstå samspillet mellem AuNP'er og biologiske systemer. Der er forskellige analytiske teknikker, herunder Fourier transform infrarød (FTIR) spektroskopi2, laser....

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1. Tænding af lasersystemet

  1. Tænd for sikringssystemet, og vælg armlaser, før du starter systemet.
  2. Tænd pc'en med softwaren til at styre femtosekundlaseren med dobbelt udgang.
  3. Indlæs softwaren til femtosekundlaseren med dobbelt udgang; Denne software gør det muligt at tænde og slukke for laseren og styrer direkte bølgelængden af pumpestrålen.
  4. Tænd for laseremissionen ved at holde tænd/sluk-ikonet nede for at tælle 3.
  5. Vent, indtil laseren er v.......

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SF-TRU-modulet (Spectral Focusing Timing and Recombination Unit) introduceres mellem femtosekundlaseren med dobbelt udgang og det modificerede laserscanningsmikroskop. Det justerbare ultrahurtige lasersystem, der anvendes i denne undersøgelse, har to udgangsporte, der leverer en stråle ved en fast bølgelængde på 1.045 nm og den anden stråle justerbar i området 680-1.300 nm. Et detaljeret skema over SF-TRU-modulet og multimodal billedbehandlingsplatform er afbildet i figur 1. SF-TRU er.......

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Denne undersøgelse har præsenteret kombinationen af SF-TRU-modul og ultrahurtigt dual-output lasersystem demonstreret sine anvendelser til multimodal mikrospektroskopi. Med sin evne til at undersøge guldnanopartiklers (AuNPs') optagelse af kræftceller kan den multimodale billeddannelsesplatform visualisere de cellulære reaktioner på hypertermiske kræftbehandlinger, når laserstråler absorberes af AuNP'er.

Desuden opnås hurtig kemisk specifik billeddannelse og høj spektralopløsning v.......

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Denne forskning blev støttet af EPSRC Grants: Raman Nanotheranostics (EP/R020965/1) og CONTRAST-facilitet (EP/S009957/1).

....

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NameCompanyCatalog NumberComments
APE SRS Detection UnitAPE (Angewandte Physik & Elektronik GmbH)APE Lock-in ModuleCombined system containing a large area Si photo-diode for detecting the pump beam along with a Lock-In amplifier for detecting the beam modulations
Confocal Scanning UnitOlympusFV 3000Confocal scanning unit used for imaging
CML Latex Beads, 4% w/v, 1.0 µmInvitrogenC37483Polystyrene microspheres
CoverslipsThorlabsCG15CH222 mm x 22 mm coverslips for seeding cells
FBSGibco10500-064Foetal Bovine Serum (Heat Inactivated)
FlouviewOlympusFV31S-SWLaser scanning microscope control software
Function GeneratorBX precision40543Used to generate square wave function which is fed to EOM in SF-TRU to produce modulations in the stokes beam
FV3000OlympusIX83P2ZFOther microscope frames can be used.
Gold NanoparticlesNanopartzA11-60Spherical gold nanoparticles, 60 nm diameter
Input Output InterfaceOlympusFV30 ANALOGThis unit allows voltage readouts from PMT and LockIn to be fed into the confocal scanning software and allows timing pulses to be sent between the olympus microscope and the SF-TRU unit.
InSight X3NewportSpectra-PhysicsDual-output femtosecond pulsed laser. Tunable (680–1300 nm) and fixed (1045 nm) laser outputs with the repetition rate of 80 MHz.
Microscope FrameOlympusIX83Inverted microscope
Mouse 4T1 cellsATCCCRL-2539Mouse breast cancer cells
NA 1.2 Water Immersion ObjectiveOlympusUPLSAPO60XW/IRThe multiphoton 60x Objective has a 0.28 mm working distance. Other similar objectives can be used.
NA 1.4 CondenserNikonCSC1003Other condensers with NA higher than the excitation objective can also be used.
PMTHamamatsuR3896PMT used for detecting anti-stokes photos for CARS micrsocopy
PMT ConnectorHamamatsuC13654-01-Y002Connector for PMT
Power SupplyRSRSPD-3303 CProgrammable power supply which is used for providing the correct voltage to the PMT
RPMI-1640GibcoA10491-01Roswell Park Memorial Institute (RPMI) 1640 Medium has since been found suitable for a variety of mammalian cells.
SF-TRUNewport Spectra PhysicsSF-TRUSystem designed for controlling the time delay and dispersion of the 2 laser outputs and for performing the beam modulations required for SRS

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