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Bioengineering

Imagem Biomolecular da Captação Celular de Nanopartículas utilizando Microscopia Óptica Multimodal Não Linear

Published: May 16th, 2022

DOI:

10.3791/63637

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

Este artigo apresenta a integração de um módulo de focalização espectral e um laser de pulso de dupla saída, permitindo imagens hiperespectrais rápidas de nanopartículas de ouro e células cancerígenas. Este trabalho tem como objetivo demonstrar os detalhes de técnicas ópticas não lineares multimodais em um microscópio de varredura a laser padrão.

Sondar nanopartículas de ouro (AuNPs) em sistemas vivos é essencial para revelar a interação entre AuNPs e tecidos biológicos. Além disso, ao integrar sinais ópticos não lineares, como espalhamento Raman estimulado (SRS), fluorescência excitada de dois fótons (TPEF) e absorção transitória (TA) em uma plataforma de imagem, ele pode ser usado para revelar o contraste biomolecular de estruturas celulares e AuNPs de maneira multimodal. Este artigo apresenta uma microscopia óptica não linear multimodal e a aplica para realizar imagens quimicamente específicas de AuNPs em células cancerígenas. Essa plataforma de imagem fornece uma nova abordagem para desenvolver AuNPs funcionalizadas mais eficientes e determinar se elas estão dentro de vasculaturas ao redor dos espaços tumoral, pericelular ou celular.

As nanopartículas de ouro (AuNPs) mostraram grande potencial como sondas de imagem biocompatíveis, por exemplo, como substratos eficazes de espectroscopia Raman aprimorada por superfície (SERS) em várias aplicações biomédicas. As principais aplicações incluem campos como biossensoriamento, bioimagem, espectroscopias aprimoradas de superfície e terapia fototérmica para o tratamento do câncer1. Além disso, sondar AuNPs em sistemas vivos é crucial para avaliar e entender a interação entre AuNPs e sistemas biológicos. Existem várias técnicas analíticas, incluindo espectroscopia de infravermelho com transformada de Fourier (FTI....

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1. Ligar o sistema a laser

  1. Ligue o sistema de intertravamento e selecione o laser do braço antes de iniciar o sistema.
  2. Ligue o PC com o software para controlar o laser de femtossegundo de saída dupla.
  3. Carregue o software para o laser de femtossegundo de saída dupla; este software permite que o laser seja ligado e desligado e controla diretamente o comprimento de onda do feixe da bomba.
  4. Ative a emissão de laser mantendo pressionado o ícone Energia para uma cont.......

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O módulo Spectral Focusing Timing and Recombination Unit (SF-TRU) é introduzido entre o laser de femtossegundo de saída dupla e o microscópio de varredura a laser modificado. O sistema de laser ultrarrápido ajustável usado neste estudo tem duas portas de saída que fornecem um feixe a um comprimento de onda fixo de 1.045 nm e o outro feixe ajustável na faixa de 680-1.300 nm. Um esquema detalhado do módulo SF-TRU e da plataforma de imagem multimodal é representado na Figura 1. O SF-T.......

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Este estudo apresentou a combinação do módulo SF-TRU e do sistema laser ultrarrápido de saída dupla demonstrando suas aplicações para microespectroscopia multimodal. Com sua capacidade de investigar a absorção de nanopartículas de ouro (AuNPs) por células cancerígenas, a plataforma de imagem multimodal pode visualizar as respostas celulares a tratamentos de câncer hipertérmico quando os feixes de laser são absorvidos por AuNPs.

Além disso, imagens quimicamente específicas ráp.......

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Esta pesquisa foi apoiada pelo EPSRC Grants: Raman Nanotheranostics (EP/R020965/1) e 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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