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.
Highlights
Single-cell biophysical assays that quantify size [1], morphology [2] and movement [3,4] of subcellular components can provide great insight into macromolecular function and effectively bridge the biological activities at the cellular and the molecular scale together [5]
In Eq (1), IP represents the pixel intensity of a particle and the fitted parameters, I ′, Ra′, μx′, and μy′ represent the particle’s peak intensity, apparent radius, and the position in the x- and y-direction of a Cartesian plane, respectively. These results indicate that the positioning error strongly depends on the EM gain setting in a manner that depended on the intensity of the tracked object. (Fig. 1)
A quantitative method to assess the outcome after using EM gain and evaluate its effectiveness in a biophysical assay is essential to ensure high resolution in subcellular measurements
Summary
Single-cell biophysical assays that quantify size [1], morphology [2] and movement [3,4] of subcellular components can provide great insight into macromolecular function and effectively bridge the biological activities at the cellular and the molecular scale together [5]. The performance of the CCD camera deteriorates with decreasing light intensity from the sample [10], and this significantly reduces the accuracy of low intensity subcellular measurements, such as in single molecule fluorescence imaging This problem can be solved by acquiring images on much more expensive avalanche photodiode detectors (APD) or photomultiplier tubes (PMT) [11,12]. 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] This capability to elevate the photon-generated signal above the readout noise of the device even at high frame rates has made it possible to meet the need for ultra-low-light imaging applications without the use of external image intensifiers [16]. We have created a general method, applicable to other types of CCD cameras, which can optimize electron multiplication for subcellular imaging, and provide a quantitative guideline to improve the accuracy of subcellular biophysical assays
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