Abstract
We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500–700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.
Highlights
The ability of fluorescence microscopy to probe intracellular processes led to the development of powerful approaches to unveil the diffusive dynamics of biomolecules
Repeated PSF scanning over a circle or a line is useful as: 1) it is equivalent of performing many spFCS measurements simultaneously, yielding a spatial map of molecular diffusivity [4], 2) it allows cross (‘‘pair’’) correlation between any two points in the line separated by n pixel [pair correlation function (pCF)(n)], ‘‘mapping’’ the routes taken by molecules from the first to the second observation point [5,6]
We initially tested the gain in spatial resolution by stimulated emission depletion (STED) over confocal imaging in our setup using immobilized YFP-labeled Capsid Like Particles (CLPs) as benchmark
Summary
The ability of fluorescence microscopy to probe intracellular processes led to the development of powerful approaches to unveil the diffusive dynamics of biomolecules. In FCS, the diffusion dynamics are recovered from the fluorescence fluctuations generated by single molecules that cross the focal volume (defined by the point spread function, or PSF) during their motion [2]. Vice-versa, in spatiotemporal FCS (stFCS) [3], the PSF is moved in a periodic pattern within the sample at a rate faster than diffusion fluctuation, introducing a spatial component into the measurements. Repeated PSF scanning over a circle or a line is useful as: 1) it is equivalent of performing many spFCS measurements simultaneously, yielding a spatial map of molecular diffusivity [4], 2) it allows cross (‘‘pair’’) correlation (pCF) between any two points in the line separated by n pixel [pCF(n)], ‘‘mapping’’ the routes taken by molecules from the first to the second observation point [5,6]
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