Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method

Byungyeon Kim; Minsuk Lee; Byungjun Park; Seungrag Lee; Youngjae Won

doi:10.1117/12.2253440

16 February 2017 Referencing techniques for high-speed confocal fluorescence lifetime imaging microscopy (FLIM) based on analog mean-delay (AMD) method

Byungyeon Kim, Minsuk Lee, Byungjun Park, Seungrag Lee, Youngjae Won

Author Affiliations +

Proceedings Volume 10068, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV; 100681Z (2017) https://doi.org/10.1117/12.2253440
Event: SPIE BiOS, 2017, San Francisco, California, United States

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.

Citation Download Citation

Byungyeon Kim, Minsuk Lee, Byungjun Park, Seungrag Lee, and 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); https://doi.org/10.1117/12.2253440

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