16 February 2017 Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method
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Abstract
Analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of lifetime to be measured. We found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. As a referencing technique, we already proposed dual-channel waveform measurement scheme that may suppress these errors. In this study, we have demonstrated real-time confocal AMD-FLIM system with dual-channel waveform measurement technique.
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Byungyeon Kim, Minsuk Lee, Byungjun Park, Seungrag Lee, Youngjae Won, "Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method", Proc. SPIE 10068, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, 100681Z (16 February 2017); doi: 10.1117/12.2253440; https://doi.org/10.1117/12.2253440
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