We present a four-wave mixing interferometry technique recently developed by us, whereby single non-fluorescing gold nanoparticles are imaged background-free even inside highly heterogeneous cellular environments, owing to their specific nonlinear plasmonic response. The set-up enables correlative four-wave mixing/confocal fluorescence imaging, opening the prospect to study the fate of nanoparticle-biomolecule-fluorophore conjugates and their integrity inside cells. Beyond imaging, the technique features the possibility to track single particles with nanometric position localization precision in 3D from rapid single-point measurements at 1 ms acquisition time, by exploiting the optical vortex field pattern in the focal plane of a high numerical aperture objective lens. These measurements are also uniquely sensitive to the particle in-plane asymmetry and orientation. The localization precision in plane is found to be consistent with the photon shot-noise, while axially it is limited to about 3nm by the nano-positioning sample stage, with an estimated photon shot-noise limit of below 1 nm. As a proof-of- principle, the axial localization is exploited to track single gold nanoparticles of 25nm radius while diffusing across aqueous pockets in a dense agarose gel, mimicking a relevant biological environment.
Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances of molecules to drive inelastic scattering of light, and thus eradicates the need for exogenous fluorescent labelling, whilst providing high-resolution three-dimensional images with chemical specificity. Replacement of hydrogen atoms with deuterium presents a labelling strategy that introduces minimal change to compound structure yet is compatible with CARS due to an induced down-shift of the CH2 peak into a region of the Raman spectrum which does not contain contributions from other chemical species, thus giving contrast against other cellular components.
We present our work using deuterated oleic acid to optimise setup of an in-house-developed multimodal, multiphoton, laser-scanning microscope for precise identification of carbon-deuterium-associated peaks within the silent region of the Raman spectrum. Application of the data analysis procedure, factorisation into susceptibilities and concentrations of chemical components (FSC3), enables the identification and quantitative spatial resolution of specific deuterated chemical components within a hyperspectral CARS image. Full hyperspectral CARS datasets were acquired from HeLa cells incubated with either deuterated or non-deuterated oleic acid, and subsequent FSC3 analysis enabled identification of the intracellular location of the exogenously applied deuterated lipid against the chemical background of the cell. Through application of FSC3 analysis, deuterium-labelling may provide a powerful technique for imaging small molecules which are poorly suited to conventional fluorescence techniques.
KEYWORDS: Nanoparticles, Signal detection, Gold, Multiphoton microscopy, Four wave mixing, Luminescence, Microscopes, Microscopy, Signal to noise ratio, Spatial resolution
We have developed a novel multiphoton microscopy technique not relying on (and hence not limited by) fluorescence
emission, which exploits the third-order nonlinearity called four-wave mixing of gold nanoparticles
in resonance with their surface Plasmon. The coherent, transient and resonant nature of this signal allows its
detection free from backgrounds that limit other contrast methods for gold nanoparticles. We show detection
of single 10nm gold nanoparticles with low excitation intensities, corresponding to negligible average thermal
heating. Owing to the the third-order nonlinearity we measure a transversal and axial resolution of 140nm
and 470nm respectively, better than the one-photon diffraction limit. We also show high-contrast imaging of
gold-labels down to 5nm size in Golgi structures of HepG2 cells at useful imaging speeds (10 kHz pixel rate).
Thermal dissociation of gold nanoparticles from their bonding sites when varying the excitation intensity is also
investigated.
We demonstrate frequency differential CARS (D-CARS) using femtosecond laser pulses linearly chirped by glass
elements of high group-velocity dispersion. By replicating the Pump-Stokes pair into a pulse train at twice the
laser repetition rate, and controlling the instantaneous frequency difference by glass dispersion, we adjust the
Raman frequency probed by each pair in an intrinsically stable way. The resulting CARS intensities are detected
simultaneously by a single photomultiplier as sum and difference using lock-in detection. We demonstrate
imaging of living cells with strongly suppressed non-resonant background. We also show D-CARS using a single
femtosecond laser source.
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