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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    • "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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    ABSTRACT: We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.
    Biomedical Optics Express 12/2010; 1(5):1408-1431. DOI:10.1364/BOE.1.001408 · 3.65 Impact Factor
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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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    ABSTRACT: Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.
    International Journal of Molecular Sciences 02/2010; 11(2):427-57. DOI:10.3390/ijms11020427 · 2.86 Impact Factor
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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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    ABSTRACT: The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 mus time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.
    Biophysics of Structure and Mechanism 08/2009; 38(6):813-28. DOI:10.1007/s00249-009-0499-9 · 2.22 Impact Factor
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