title: Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.
abstract: Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic.
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology , University of Liverpool , Biosciences Building, Crown Street, Liverpool L69 7ZB, UK ; EMBL Australia Node in Single Molecule Science, Lowy Cancer Research Centre , University of New South Wales , Sydney, NSW 2052, Australia.
authors.forename: D J
authors.initials: DJ
authors.lastname: Nieves
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology , University of Liverpool , Biosciences Building, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology , University of Liverpool , Biosciences Building, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: D G
authors.initials: DG
authors.lastname: Fernig
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology , University of Liverpool , Biosciences Building, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: R
authors.initials: R
authors.lastname: Lévy
keywords: FGF2; PHI; RICS; diffusion; fluctuation spectroscopy; gold nanoparticles