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

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.

High-throughput time-correlated single photon counting.

We demonstrate time-correlated single photon counting (TCSPC) in microfluidic droplets under high-throughput conditions. We discuss the fundamental limitations in the photon acquisition rate imposed by the single photon detection technique and show that it does not preclude accurate fluorescence lifetime (FLT) measurements at a droplet throughput exceeding 1 kHz with remarkable sensitivity. This work paves the way for the implementation of innovative biomolecular interaction assays relying on the FLT detection of nanosecond-lived fluorophores for high-throughput biotechnological applications, including high-throughput screening or cell sorting potentially allowed by droplet microfluidics or other fast sample handling facilities.