22 February 2011 Flow cytometric separation of spectrally overlapping fluorophores using multifrequency fluorescence lifetime analysis
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Abstract
Digital excited state lifetime measurements in cytometry were performed on multi-tagged Chinese Hamster Ovary (CHO) cells in order to discriminate between spectrally overlapping fluorescent species. Fluorescence lifetime was determined through digital Fourier analysis with a specialized data acquisition system subsequent to multi-frequency intensity modulation by a solid-state laser excitation source. This work demonstrates that square wave modulation coupled with digital lifetime signal processing can lead to separation of ethidium bromide (EB) and propidium iodide (PI), in cells stained with both dyes. By driving the square wave modulation of the laser at 2 MHz, we were able to access the multiple harmonics present within that wave. In an offline analysis, the phase differences of scatter and fluorescence channels were examined at each harmonic of the primary frequency. The phase difference revealed approximate fluorescence lifetimes of 27.1-ns and 13.0-ns for the EB and PI, respectively. Although the absolute lifetime of each species was not resolved to high accuracy, this work shows a clear separation of the lifetime value calculated at each harmonic. The calculated values that most closely corresponded to the single-dye and multiple-dye average lifetimes were found at the fundamental harmonic frequency (2 MHz) as well as the 4th harmonic (14MHz) frequency. At 2 and 14MHz the average lifetime was 27.1ns and 13.0ns, respectively.
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Patrick L. Jenkins, Patrick L. Jenkins, James P. Freyer, James P. Freyer, Mark S. Naivar, Mark S. Naivar, Alexandra Arteaga, Alexandra Arteaga, Jessica P. Houston, Jessica P. Houston, } "Flow cytometric separation of spectrally overlapping fluorophores using multifrequency fluorescence lifetime analysis", Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 790216 (22 February 2011); doi: 10.1117/12.875627; https://doi.org/10.1117/12.875627
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