Analysis of Molecular Concentration and Brightness from Fluorescence Fluctuation Data with an Electron Multiplied CCD Camera

Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine, California 92697, USA.
Biophysical Journal (Impact Factor: 3.97). 10/2008; 95(11):5385-98. DOI: 10.1529/biophysj.108.130310
Source: PubMed


We demonstrate the calculation of particle brightness and concentration from fluorescence-fluctuation photon-counting statistics using an electron-multiplied charge-coupled device (EMCCD) camera. This technique provides a concentration-independent measure of particle brightness in dynamic systems. The high sensitivity and highly parallel detection of EMCCD cameras allow for imaging of dynamic particle brightness, providing the capability to follow aggregation reactions in real time. A critical factor of the EMCCD camera is the presence of nonlinearity at high intensities. These nonlinearities arise due to limited capacity of the CCD well and to the analog-to-digital converter maximum range. However, we show that the specific camera we used (with a 16-bit analog-to-digital converter) has sufficient dynamic range for most microscopy applications. In addition, we explore the importance of camera timing behavior as it is affected by the vertical frame transfer speed of the camera. Although the camera has microsecond exposure time for illumination of a few pixels, the exposure time increased to milliseconds for full-field illumination. Finally, we demonstrate the ability of the technique to follow concentration changes and measure single-molecule brightness in real time in living cells.

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Available from: Jay R Unruh, Oct 01, 2014
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    • "Several intensity-enhanced techniques have been developed to obtain high contrast images from low intensity signals such as intensified CCD (ICCD) and electron multiplying CCD (EMCCD) [10]. After the back-illuminated EMCCD camera was introduced, the sensitivity of EMCCD has outperformed the ICCD and the EMCCD has become a popular choice for imaging the dynamics of single molecules in cells [13,14]. EMCCD utilizes several specialized extended serial registers on the CCD chip to apply a high voltage and produce multiplying gain through the process of impact ionization in silicon [15]. "
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    ABSTRACT: The electron-multiplying charge-coupled device (EMCCD) camera possesses an electron multiplying function that can effectively convert the weak incident photon signal to amplified electron output, thereby greatly enhancing the contrast of the acquired images. This device has become a popular photon detector in single-cell biophysical assays to enhance subcellular images. However, the quantitative relationship between the resolution in such measurements and the electron multiplication setting in the EMCCD camera is not well-understood. We therefore developed a method to characterize the exact dependence of the signal-to-noise-ratio (SNR) on EM gain settings over a full range of incident light intensity. This information was further used to evaluate the EMCCD performance in subcellular particle tracking. We conclude that there are optimal EM gain settings for achieving the best SNR and the best spatial resolution in these experiments. If it is not used optimally, electron multiplication can decrease the SNR and increases spatial error.
    Optics Express 03/2010; 18(5):5199-212. DOI:10.1364/OE.18.005199 · 3.49 Impact Factor
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    • "The number and brightness (N&B) approach to image analysis was recently introduced by Enrico Gratton's laboratory (Digman et al. 2008b, 2009). This technique can be applied to images acquired using confocal microscopy or TIRF (Unruh and Gratton 2008) as long as the pixel dwell time is less than the characteristic diffusion time of the particle. The N&B approach can be considered the imaging equivalent of the PCH method; however, N&B does not require a non-linear fit of the data, and the average particle number <N> and particle brightness B are extracted directly from the image intensity data. "
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    ABSTRACT: Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature.
    Biophysical Reviews 09/2009; 1(3):105-118. DOI:10.1007/s12551-009-0013-8

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