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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.
Highlights
The direct observation of individual molecules by optical microscopy [1] in living cells [2,3,4,5,6] allows access to movement, fluctuations, colocalization and conformational changes at the2015 The Authors
Photothermal heterodyne imaging (PHI) imaging with a pixel dwell time of 80 μs has been achieved recently using a galvanometric laser scanning system [52]
This signal-to-noise ratio (SNR) is high enough to be used for single nanoparticle tracking [34], the acquisition of rapid raster scan images with a piezo-stage PHI microscope for Raster image correlation spectroscopy (RICS) analysis is possible
Summary
The direct observation of individual molecules by optical microscopy [1] in living cells [2,3,4,5,6] allows access to movement, fluctuations, colocalization and conformational changes at the2015 The Authors. One example is the organization of the cell membrane gained by SPT of transmembrane receptors labelled with gold nanoparticles [9,10], e.g. the compartmentalization of the cell membrane, as a mechanism to modulate membrane movement and function [9,10]. Both SMT and SPT, to effectively probe the biological system, require a large number of tracks to provide statistical power to the measurements [17,18,20]. To improve coverage over larger areas on single cells within a single experiment, Giannone et al [20] introduced universal point accumulation imaging in the nanoscale topography (uPAINT), which uses constant imaging during cell labelling to generate a super-resolution image based on short (before photobleaching) single molecule tracks
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