Biomolekylær billeddannelse af cellulær optagelse af nanopartikler ved hjælp af multimodal ikke-lineær optisk mikroskopi
Summary
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.
Abstract
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.
Introduction
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, laserablation induktivt koblet massespektrometri (LA-ICP-MS)3 og magnetisk resonansbilleddannelse (MRI)4, der med succes er blevet brugt til at undersøge fordelingen af AuNP’er i væv. Ikke desto mindre lider disse metoder af flere ulemper, såsom at være tidskrævende og involvere kompleks prøveforberedelse3, der kræver lange anskaffelsestider, eller manglen på submikron rumlig opløsning 2,4.
Sammenlignet med konventionelle billeddannelsesteknikker giver ikke-lineær optisk mikroskopi flere fordele ved sondering af levende celler og AuNP’er: Den ikke-lineære optiske mikroskopi opnår dybere billeddybde og giver iboende 3D optisk sektionsfunktion ved brug af nær-IR ultrahurtige lasere. Med den betydelige forbedring af billeddannelseshastighed og detektionsfølsomhed er to-foton exciteret fluorescens (TPEF) 5,6,7 og anden harmoniske generation (SHG)8,9,10 mikroskopi blevet demonstreret for yderligere at forbedre ikke-invasiv billeddannelse af endogene biomolekyler i levende celler og væv. Desuden er det ved hjælp af nye pumpesonde ikke-lineære optiske teknikker såsom forbigående absorption (TA) 11,12,13,14 og stimuleret Raman-spredning (SRS)15,16,17,18 muligt at udlede etiketfri biokemisk kontrast mellem cellulære strukturer og AuNP’er. Visualisering af AuNP’er uden brug af ydre etiketter er af stor betydning, da kemiske forstyrrelser af nanopartiklerne vil ændre deres fysiske egenskaber og dermed deres optagelse i celler.
Denne protokol præsenterer implementeringen af et SF-TRU-modul (Spectral Focusing Timing and Recombination Unit) til en pulslaser med dobbelt bølgelængde, der muliggør hurtig multimodal billeddannelse af AuNP’er og kræftceller. Dette arbejde har til formål at demonstrere detaljerne i integrerede TPEF-, TA- og SRS-teknikker på et laserscanningsmikroskop.
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af EPSRC Grants: Raman Nanotheranostics (EP/R020965/1) og CONTRAST-facilitet (EP/S009957/1).
Materials
| APE SRS Detection Unit | APE (Angewandte Physik & Elektronik GmbH) | APE Lock-in Module | Combined 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 Unit | Olympus | FV 3000 | Confocal scanning unit used for imaging |
| CML Latex Beads, 4% w/v, 1.0 µm | Invitrogen | C37483 | Polystyrene microspheres |
| Coverslips | Thorlabs | CG15CH2 | 22 mm x 22 mm coverslips for seeding cells |
| FBS | Gibco | 10500-064 | Foetal Bovine Serum (Heat Inactivated) |
| Flouview | Olympus | FV31S-SW | Laser scanning microscope control software |
| Function Generator | BX precision | 40543 | Used to generate square wave function which is fed to EOM in SF-TRU to produce modulations in the stokes beam |
| FV3000 | Olympus | IX83P2ZF | Other microscope frames can be used. |
| Gold Nanoparticles | Nanopartz | A11-60 | Spherical gold nanoparticles, 60 nm diameter |
| Input Output Interface | Olympus | FV30 ANALOG | This 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 X3 | Newport | Spectra-Physics | Dual-output femtosecond pulsed laser. Tunable (680–1300 nm) and fixed (1045 nm) laser outputs with the repetition rate of 80 MHz. |
| Microscope Frame | Olympus | IX83 | Inverted microscope |
| Mouse 4T1 cells | ATCC | CRL-2539 | Mouse breast cancer cells |
| NA 1.2 Water Immersion Objective | Olympus | UPLSAPO60XW/IR | The multiphoton 60x Objective has a 0.28 mm working distance. Other similar objectives can be used. |
| NA 1.4 Condenser | Nikon | CSC1003 | Other condensers with NA higher than the excitation objective can also be used. |
| PMT | Hamamatsu | R3896 | PMT used for detecting anti-stokes photos for CARS micrsocopy |
| PMT Connector | Hamamatsu | C13654-01-Y002 | Connector for PMT |
| Power Supply | RS | RSPD-3303 C | Programmable power supply which is used for providing the correct voltage to the PMT |
| RPMI-1640 | Gibco | A10491-01 | Roswell Park Memorial Institute (RPMI) 1640 Medium has since been found suitable for a variety of mammalian cells. |
| SF-TRU | Newport Spectra Physics | SF-TRU | System designed for controlling the time delay and dispersion of the 2 laser outputs and for performing the beam modulations required for SRS |
Referências
- Tabish, T. A., et al. Smart gold nanostructures for light mediated cancer theranostics: Combining optical diagnostics with photothermal therapy. Advanced Science. 7 (15), 1903441 (2020).
- Tian, F., et al. Gold nanostars for efficient in vitro and in vivo real-time SERS detection and drug delivery via plasmonic-tunable Raman/FTIR imaging. Biomaterials. 106, 87-97 (2016).
- Jenkins, S. V., et al. Enhanced photothermal treatment efficacy and normal tissue protection via vascular targeted gold nanocages. Nanotheranostics. 3 (2), 145-155 (2019).
- Huang, J., et al. Rational design and synthesis of gammaFe2 O3 @Au magnetic gold nanoflowers for efficient cancer theranostics. Advanced Materials. 27 (34), 5049-5056 (2015).
- Dilipkumar, A., et al. Label-free multiphoton endomicroscopy for minimally invasive in vivo imaging. Advanced science. 6 (8), 1801735 (2019).
- Wang, C. -. C., et al. Differentiation of normal and cancerous lung tissues by multiphoton imaging. Journal of Biomedical Optics. 14 (4), 044034 (2009).
- Chrabaszcz, K., et al. Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer. The Analyst. 145 (14), 4982-4990 (2020).
- Tsai, T. H., et al. Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo. Journal of Dermatological Science. 65 (2), 95-101 (2012).
- Wang, C. -. C., et al. Early development of cutaneous cancer revealed by intravital nonlinear optical microscopy. Applied Physics Letters. 97 (11), 113702 (2010).
- Li, F. -. C., et al. Dorsal skin fold chamber for high resolution multiphoton imaging. Optical and Quantum Electronics. 37 (13), 1439-1445 (2005).
- Tong, L., et al. Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy. Nature Nanotechnology. 7 (1), 56-61 (2011).
- Chong, S., Min, W., Xie, X. S. Ground-state depletion microscopy: Detection sensitivity of single-molecule optical absorption at room temperature. The Journal of Physical Chemistry Letters. 1 (23), 3316-3322 (2010).
- Chen, T., et al. Transient absorption microscopy of gold nanorods as spectrally orthogonal labels in live cells. Nanoscale. 6 (18), 10536-10539 (2014).
- Liu, J., Irudayaraj, J. M. Non-fluorescent quantification of single mRNA with transient absorption microscopy. Nanoscale. 8 (46), 19242-19248 (2016).
- Freudiger, C. W., et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science. 322 (5909), 1857-1861 (2008).
- Wang, C. -. C., et al. In situ chemically specific mapping of agrochemical seed coatings using stimulated Raman scattering microscopy. Journal of Biophotonics. 11 (11), 201800108 (2018).
- Wang, C. -. C., Yoong, F. -. Y., Penfield, S., Moger, J. Visualization of active ingredients uptake in seed coats with stimulated Raman scattering microscopy. Proceedings SPIE 10069, Multiphoton Microscopy the Biomedical Sciences XVII. , 1006928 (2017).
- Hu, F., Shi, L., Min, W. Biological imaging of chemical bonds by stimulated Raman scattering microscopy. Nature Methods. 16 (9), 830-842 (2019).
- Zeytunyan, A., Baldacchini, T., Zadoyan, R. Module for multiphoton high-resolution hyperspectral imaging and spectroscopy. Proceedings SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII. , 104980 (2018).
- Wang, C. -. C., Wu, R. -. J., Lin, S. -. J., Chen, Y. -. F., Dong, C. -. Y. Label-free discrimination of normal and pulmonary cancer tissues using multiphoton fluorescence ratiometric microscopy. Applied Physics Letters. 97 (4), 043706 (2010).
- Wang, C. -. C., Chandrappa, D., Smirnoff, N., Moger, J. Monitoring lipid accumulation in the green microalga botryococcus braunii with frequency-modulated stimulated Raman scattering. Proceedings SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV. , 9329 (2015).
- Figueroa, B., et al. Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source. Biomedical Optics Express. 9 (12), 6116-6131 (2018).
- Cui, L., et al. In situ plasmon-enhanced CARS and TPEF for Gram staining identification of non-fluorescent bacteria. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 264, 120283 (2022).
- Ma, J., Sun, M. Nonlinear optical microscopies (NOMs) and plasmon-enhanced NOMs for biology and 2D materials. Nanophotonics. 9 (6), 1341-1358 (2020).
- Sun, L., Chen, Y., Sun, M. Exploring nonemissive excited-state intramolecular proton transfer by plasmon-enhanced hyper-Raman scattering and two-photon excitation fluorescence. The Journal of Physical Chemistry C. 126 (1), 487-492 (2022).
