Four-wave-mixing microscopy reveals non-colocalisation between gold nanoparticles and fluorophore conjugates inside cells

Abstract

Gold nanoparticles have been researched for many biomedical applications in diagnostics, theranostics, and as drug delivery systems. When conjugated to fluorophores, their interaction with biological cells can be studied in situ and real time using fluorescence microscopy. However, an important question that has remained elusive to answer is whether the fluorophore is a faithful reporter of the nanoparticle location.Here, our recently developed four-wave-mixing optical microscopy is applied to image individual gold nanoparticles and in turn investigate their co-localisation with fluorophores inside cells. Nanoparticles from 10 nm to 40 nm diameter were conjugated to fluorescently-labeled transferrin, for internalisation via clathrin-mediated endocytosis, or to non-targeting fluorescently-labelled antibodies. Human (HeLa) and murine (3T3-L1) cells were imaged at different time points after incubation with these conjugates. Our technique identified that, in most cases, fluorescence originated from unbound fluorophores rather than from fluorophores attached to nanoparticles. Fluorescence detection was also severely limited by photobleaching, quenching and autofluorescence background. Notably, correlative extinction/fluorescence microscopy of individual particles on a glass surface indicated that commercial constructs contain large amounts of unbound fluorophores. These findings highlight the potential problems of data interpretation when reliance is solely placed on the detection of fluorescence within the cell, and are of significant importance in the context of correlative light electron microscopy. † Electronic supplementary information (ESI) available: Fig. S1-S17. See

Green synthesis of glyco-CuInS2 QDs with visible/NIR dual emission for 3D multicellular tumor spheroid and in vivo imaging

“…In view of this, the synthesis of cell membrane-targeted fluorescent bioprobes is of great importance [ 57 ]. Furthermore, in order to determine whether the fluorescence emitted by the two channels remains in the same position, a fluorescence co-localization assay was performed [ 58 ]. From the scattering trend of pixel points in the co-localized pixel plots in Fig.…”

Correlative light electron microscopy using small gold nanoparticles as single probes

“…FWM set-up. FWM microscopy was performed using a home built set-up, as described in detail in our recent works [7,15]. Briefly, optical pulses of 150 fs duration centered at 550 nm wavelength with ν L =80 MHz repetition rate were provided by the signal output of an optical parametric oscillator (Spectra Physics Inspire HF 100) pumped by a frequency-doubled femtosecond Ti:Sa laser (Spectra Physics Mai Tai HP).…”

“…Indeed, fluorescence quenching, due to nonradiative transfer in the vicinity of a AuNP, is a well documented effect, which can significantly reduce the applicability of these probes in CLEM workflows [5,6]. Moreover, we have shown recently that the integrity of this type of dual probes inside cells, and in turn their ability to correlatively report the location of the same molecule, should be seriously questioned [7].…”

“…Recently, we developed a multiphoton LM technique which exploits the fourwave mixing (FWM) nonlinearity of AuNPs, triply resonant to the LSPR. With this method we were able to detect individual small (down to 5 nm radius) AuNPs inside scattering cells [7,15] and tissues [16] completely free from background, at imaging speeds and excitation powers compatible with live cell imaging, with a sensitivity limited only by photon shot noise.…”

Roadmap on bio-nano-photonics

“…Parallelisation of acquisition (multiple scan points) and optimisation of acquisition are now the targets of development to allow the required fast timelapse imaging at nm resolution in live cells required to understand the dynamics of membrane trafficking. Orientation of nanoparticle cargo and their dynamics within vesicles can be studied using parallax-quantified differential interference contrast microscopy [49], dual colour dual particle tracking [50] and four-wave-mixing microscopy [51,52]. Although spatial (10 s of nm) and temporal (ms) resolution of these techniques is excellent, the sensitivity of the systems still require relatively large (compared to proteins) gold spheres, nanorods or fluorescently labelled latex beads.…”