Abstract
The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.
Highlights
The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution
Fluorescence fluctuation techniques have been employed in biophysical research for many years[1,2,3,4,5,6,7] and, in the past decade, several new concepts and implementations of fluctuation spectroscopy were introduced in order to answer specific questions regarding complex dynamic behaviors in cellular systems
FCS has been used with super-resolution techniques, such as stimulated emission depletion[17,18,19] (STED) and separation of photons by lifetime tuning[1,20,21] (SPLIT), achieving a size of the illumination volume of ~80 nm for EGFP diffusing in living cells
Summary
The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. 1234567890():,; Fluorescence fluctuation techniques have been employed in biophysical research for many years[1,2,3,4,5,6,7] and, in the past decade, several new concepts and implementations of fluctuation spectroscopy were introduced in order to answer specific questions regarding complex dynamic behaviors in cellular systems Techniques such as the pair correlation function[8,9,10,11] (pCF and 2D-pCF) are capable of detecting diffusion barriers and molecular connectivity, while number and brightness analysis[12] (N&B) can be used for mapping concentration and oligomerization states of the diffusing probe. Fluorescence fluctuation techniques are generally implemented one at a time, due to the need for a dedicated setup (e.g. spot-variation FCS), a fast temporal sampling (e.g. FCS) or a spatial sampling (e.g. pCF, iMSD)
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