Abstract
Fluorescence (cross-)correlation spectroscopy (FCS/FCCS) and generally fluorescence fluctuation spectroscopy (FFS) are confocal microscopy-based methods that allow to assess diffusion and transport properties as well as interactions of molecules (proteins, nucleic acids, compounds) in vitro and in vivo. Commercially available instrumentation enables routine measurements at one or few specific points inside living cells. However, conventional FCS/FCCS experiments remain challenging because point measurements in a living cell are associated with large error caused by the heterogeneous environment of the cellular interior. Moreover, biological noise due to cell-to-cell variations of physical and biological parameters (e.g. intracellular viscosity, protein expression levels) induces further variations, which are difficult to separate from the measurement error. Currently, these problems are partially addressed by performing statistical data analysis of measurements from many different cells. However, it is desirable to obtain more reliable and robust data from single cells with spatial resolution. This requires a new approach allowing to perform simultaneous measurements and to circumvent the problems associated with confocal FFS: photobleaching, out-of-focus illumination and loss of spatial definition due to cell movements. Here, we present a novel microscope that allows spatially resolved FFS measurements in 2D optical sections across cells. The setup is based on a single plane illumination microscope in which a thin diffraction-limited light sheet is used to illuminate a cross-section of the cell. The use of an electron-multiplying charge-coupled device (EM-CCD), placed perpendicular to the light sheet, with hundreds of single pixel detectors instead of an avalanche photodiode (a single pixel detector) enables to record on each pixel the incoming photons with single photon sensitivity and sub-millisecond time resolution. This is predicted to significantly reduce the error associated with single point measurements. It should also provide access to spatially resolved measurements of concentrations, interactions and mobilities.
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