ABSTRACT
Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.
ABSTRACT
Articular cartilage extracellular matrix imposes a significant transport barrier to albumin, the principal carrier of fatty acids. It has not been previously established whether it also influences the transport of fatty acids important for chondrocyte metabolism. Albumin was labelled with rhodamine-maleimide and bound to NBD-labelled lauric acid. Plugs of fresh equine metacarpal-phalangeal cartilage and subchondral bone were incubated with the complex at 4 degrees C for 2-160 h. The fluorophore distribution was quantified using quantitative microscopy in histological sections. The fluorescence intensity of both fluorophores fell steeply over 300 microm below the articular surface and remained relatively uniform through the mid zone but the ratio of lauric acid to albumin was higher than in the incubation medium. The effective diffusivity of lauric acid in the mid zone was (2.2+/-0.7) x 10(-12) m2 s(-1) (n = 33), higher than that of the carrier albumin, suggesting dissociation in the surface layer. Lauric acid accumulated reversibly at the tidemark.
