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


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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    • "This new technique was introduced by Celli et al., [64] [65] and combines the GP methodology with fluorescence correlation spectroscopy (FCS). FCS methods (e.g., [66] [67] [68] [69] [70] [71] [72]) are based on the analysis of intensity fluctuations caused by fluorescently labeled molecules moving through the small observation volume of a confocal or two-photon excitation microscope. Since the temporal window of these fluctuations is given by the processes determining the mobility of the molecules and their photophysics, this technique has been extensively applied to study diffusion, transport, chemical reactions, etc. (reviewed in [73]). "
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    • "In contrast, conventional laser-scanning microscopy provides spatial information about the time-averaged fluorescence intensity, but is not as well suited for picking up temporal changes. One variant of FCS that can provide spatial information is parallel multispot FCS, which originally used two parallel detection volumes (Brinkmeier et al. 1999) and has more recently been extended by using charge-coupled device detectors (Burkhardt and Schwille 2006; Kannan et al. 2006). Another modification to FCS that makes it possible to obtain both spatial and temporal information is to scan the beam (or the sample) in a linear, a circular, or a random fashion (scanning fluorescence correlation spectroscopy [sFCS]). "
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    • "Past approaches trying to use several SPADs were limited by cost and bulkiness, but also revealed the critical need of obtaining close to perfect multi-spot excitation patterns matching the detector arrangements [6,7]. More recently, signal detection using ultrasensitive cameras has been proposed, using either a confocal excitation scheme [8,9] or a widefield excitation scheme [10–12]. Although promising, these approaches have limited temporal resolution due to the finite frame rate of current cameras and are therefore limited to slow diffusion processes such as those encountered in live cells. "
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