Article

Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy

Technische Universität Dresden, Dresden, Saxony, Germany
Optics Express (Impact Factor: 3.49). 07/2006; 14(12):5013-20. DOI: 10.1364/OE.14.005013
Source: PubMed

ABSTRACT Fluorescence correlation spectroscopy (FCS) is carried out with an electron multiplying CCD (EMCCD). This new strategy is compared to standard detection by an avalanche photo diode showing good agreement with respect to the resulting autocorrelation curves. Applying different readout modes, a time resolution of 20 micros can be achieved, which is sufficient to resolve the diffusion of free dye in solution. The advantages of implementing EMCCD cameras in wide-field ultra low light imaging, as well as in multi-spot confocal laser scanning microscopy, can consequently also be exploited for spatially resolved FCS. First proof-of-principle FCS measurements with two excitation volumes demonstrate the advantage of the flexible CCD area detection.

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Available from: Petra Schwille, Sep 22, 2014
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    • "The signal from the two foci can be, then, simultaneously detected by different pixels of an electron multiplying charge-coupled device (EMCCD) placed in the image plane of the microscope. Although it is possible to detect the signal from the two foci using a dual-core optical fibre and two single point detectors [102], the setup based on an EMCCD is more flexible; it allows changing the interfocal distance (provided that a sufficient spacing between the pixels detecting signal from the two foci is maintained to minimize the crosstalk between them) and the same detection scheme is suitable also for measurements in more than two points simultaneously in order to perform cross-correlations between more pairs of foci [103–105]. Another experimentally simple method of 2-focus FCS uses a standard single focus laser scanning microscope and alternate scanning of two parallel lines [26,34,97]. "
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    • "(4) Imaging of molecular dynamics can be performed by parallelized FCS data acquisition using multifocal systems, which combine two-dimensional imaging and optical sectioning with FCS in one or more dimensions. Examples of this approach are double-focus FCS (dfFCS; Bayer and Radler 2006; Brinkmeier et al. 1997; Burkhardt and Schwille 2006; Hwang and Wohland 2007; Lumma et al. 2003; Pan et al. 2007), multifocal spinning disk FCS (Sisan et al. 2006), and total internal reflection fluorescence correlation microscopy (TIR-FCM; Kannan et al. 2007; Schwille 2003). The parallelized FCS measurements reduce the data acquisition time significantly. "
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