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Bioengineering

Biomolekylær avbildning av cellulær opptak av nanopartikler ved bruk av 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 artikkelen presenterer integrasjonen av en spektralfokuseringsmodul og en dual-output pulslaser, som muliggjør rask hyperspektral avbildning av gull nanopartikler og kreftceller. Dette arbeidet tar sikte på å demonstrere detaljene i multimodale ikke-lineære optiske teknikker på et standard laserskanningsmikroskop.

Sondering av gull nanopartikler (AuNPs) i levende systemer er viktig for å avsløre samspillet mellom AuNPs og biologiske vev. Videre, ved å integrere ikke-lineære optiske signaler som stimulert Raman-spredning (SRS), to-foton eksitert fluorescens (TPEF) og forbigående absorpsjon (TA) i en bildeplattform, kan den brukes til å avsløre biomolekylær kontrast av cellulære strukturer og AuNPs på en multimodal måte. Denne artikkelen presenterer en multimodal ikke-lineær optisk mikroskopi og bruker den til å utføre kjemisk spesifikk avbildning av AuNPs i kreftceller. Denne bildebehandlingsplattformen gir en ny tilnærming for å utvikle mer effektive funksjonaliserte AuNPs og avgjøre om de er innenfor vaskulaturer som omgir svulsten, pericellulære eller cellulære rom.

Gull nanopartikler (AuNPs) har vist stort potensial som biokompatible bildebehandlingsprober, for eksempel som effektive overflateforbedrede Raman-spektroskopi (SERS) substrater i ulike biomedisinske applikasjoner. Store applikasjoner inkluderer felt som biosensing, bioimaging, overflateforbedrede spektroskopier og fototermisk terapi for kreftbehandling1. Videre er sondering av AuNPs i levende systemer avgjørende for å vurdere og forstå samspillet mellom AuNPs og biologiske systemer. Det finnes ulike analytiske teknikker, inkludert Fourier transform infrared (FTIR) spektroskopi2, laserablasjon induk....

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1. Slå på lasersystemet

  1. Slå på låsesystemet og velg armlaser før du starter systemet.
  2. Slå på PC-en med programvaren for å kontrollere femtosekundlaseren med dobbel utgang.
  3. Last inn programvaren for femtosekundlaseren med dobbel utgang; Denne programvaren gjør det mulig å slå laseren på og av og styrer bølgelengden til pumpestrålen direkte.
  4. Slå på laserutslippet ved å holde nede på strømikonet for en telling på 3.
  5. Vent til laseren har .......

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Spectral Focusing Timing and Recombination Unit (SF-TRU)-modulen introduseres mellom femtosekundlaseren med dobbel utgang og det modifiserte laserskanningsmikroskopet. Det justerbare ultraraske lasersystemet som brukes i denne studien har to utgangsporter som leverer en stråle ved en fast 1,045 nm bølgelengde og den andre strålen som er justerbar i området 680-1,300 nm. Et detaljert skjema av SF-TRU-modulen og multimodal bildebehandlingsplattform er avbildet i figur 1. SF-TRU brukes til .......

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Denne studien har presentert kombinasjonen av SF-TRU-modul og ultrafast dual-output lasersystem demonstrerte sine applikasjoner for multimodal mikrospektroskopi. Med sin evne til å undersøke gull nanopartikler '(AuNPs) opptak av kreftceller, kan den multimodale bildebehandlingsplattformen visualisere cellulære responser på hypertermiske kreftbehandlinger når laserstråler absorberes av AuNPs.

Videre oppnås rask kjemisk spesifikk avbildning og høy spektral oppløsning ved å bruke spektr.......

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Denne forskningen ble støttet av EPSRC Grants: Raman Nanotheranostics (EP / R020965 / 1) og CONTRAST facility (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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