Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Papkovsky, Dmitri B.; Okkelman, Irina A.; Dmitriev, Ruslan I.; Foley, Tara

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

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Published Version

Date

2016-12-14

Authors

Papkovsky, Dmitri B.

Okkelman, Irina A.

Dmitriev, Ruslan I.

Foley, Tara

Papkovsky, Dmitri B.

Publisher

Public Library of Science

Abstract

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Keywords

Organoids , Cell cycle and cell division , Cell staining , Fluorescence imaging , Synthesis phase , Gastrointestinal tract , Cell proliferation , Fluorescence microscopy

Citation

Okkelman, I. A., Dmitriev, R. I., Foley, T. and Papkovsky, D. B. (2016) 'Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase', PLOS ONE, 11(12), pp. e0167385. doi:10.1371/journal.pone.0167385