AiryScan Comprehensive Super-Resolution Correlation Analysis

Abstract

AiryScan is a detector array developed by Zeiss that consists in 32 GaAs PMT detectors arranged in a hexagonal pattern. The development of single point detector arrays is recently increasing also thanks to the possibility of achieving super-resolution without signal loss by means of Image Scanning Microscopy (ISM, Sheppard et al. 2013). Furthermore, super-resolution techniques applied to fluorescence correlation techniques have shown to be effective in retrieving information that is not accessible with diffraction-limited methods, giving new valuable tools to biological research. Being essentially a high-speed nano-camera, AiryScan allows, in principle, for the implementation of most of the state-of-the-art correlation techniques at super-resolution in a single analysis. In this work we will present the parallel implementation of fluorescence correlation spectroscopy (FCS, Digman et al. 2011), pair-correlation function (pCF, Hinde et al. 2010) and connectivity analysis, image-derived mean square displacement (iMSD, Di Rienzo et al. 2014), number and brightness (N&B, Digman et al. 2008) and diffusion law analysis (Wawrezinieck et al. 2005) in the same single-spot dataset in the cellular environment. This pipeline ultimately returns information about diffusion, concentration, environment structure and accessibility, spatiotemporal confinement and oligomerization states from a single analysis of a few seconds in living cells.The combination of fast sampling speed (down to 1.23 µs per frame) and super-resolution capability opens the doors to the investigation of spatiotemporal dynamics that are not accessible with any other method, at present. We will demonstrate the possibility and the advantages of having access to several methods in a single acquisition and will show several examples of application in live cells, for instance by analyzing EGFP diffusion in NIH-3T3 cells.