ABSTRACT
Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the 'super-resolution' limit and what opportunities remain for further improvements in the near future.
Subject(s)
Limit of Detection , Microscopy/trends , Animals , Humans , Microscopy/methods , Microscopy, Fluorescence/methods , Microscopy, Fluorescence/trends , Scattering, RadiationABSTRACT
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.
ABSTRACT
A single maleimide was installed onto the self-assembled monolayer of gold nanoparticles by copper-free click chemistry. Simple covalent biofunctionalisation is demonstrated by coupling fibroblast growth factor 2 and an oligosaccharide in a 1 : 1 stoichiometry by thiol-Michael addition.
