Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

Xiao Peng; Danying Lin; Yan Wang; Jing Qi; Wei Yan; Junle Qu

doi:10.1117/12.2075075

2 March 2015 Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

Xiao Peng, Danying Lin, Yan Wang, Jing Qi, Wei Yan, Junle Qu

Author Affiliations +

Proceedings Volume 9328, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII; 93281U (2015) https://doi.org/10.1117/12.2075075
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

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Xiao Peng, Danying Lin, Yan Wang, Jing Qi, Wei Yan, and Junle Qu "Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy", Proc. SPIE 9328, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XIII, 93281U (2 March 2015); https://doi.org/10.1117/12.2075075

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KEYWORDS

Luminescence

Fluorescence lifetime imaging

Green fluorescent protein

Polymers

Microscopy

Proteins

Molecular interactions

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