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Open AccessArticle

Application of Fluorescence Lifetime Imaging Microscopy of DNA Binding Dyes to Assess Radiation-Induced Chromatin Compaction Changes

1
Department of Biophysics, GSI Helmholzzentrum für Schwerionenforschung GmbH, 64291 Darmstadt, Germany
2
Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(8), 2399; https://doi.org/10.3390/ijms19082399
Received: 2 July 2018 / Revised: 2 August 2018 / Accepted: 10 August 2018 / Published: 14 August 2018
(This article belongs to the Special Issue Advances and Challenges in Biomolecular Radiation Research)
In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions—but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for the inspection of the molecular environment of fluorophores in living cells. Here, we present the use of single organic minor groove DNA binder dyes in FLIM for measuring chromatin changes following modulation of chromatin structure in living cells. Treatment with histone deacetylase inhibitors led to an increased fluorescence lifetime indicating global chromatin decompaction, whereas hyperosmolarity decreased the lifetime of the used dyes, thus reflecting the expected compaction. In addition, we demonstrate that time domain FLIM data based on single photon counting should be optimized using pile-up and counting loss correction, which affect the readout even at moderate average detector count rates in inhomogeneous samples. Using these corrections and utilizing Hoechst 34580 as chromatin compaction probe, we measured a pan nuclear increase in the lifetime following irradiation with X-rays in living NIH/3T3 cells thus providing a method to measure radiation-induced chromatin decompaction. View Full-Text
Keywords: FLIM microcopy; Hoechst 34580; Syto 13; chromatin compaction; histone deacetylation inhibitor (HDACi); irradiation; pile-up FLIM microcopy; Hoechst 34580; Syto 13; chromatin compaction; histone deacetylation inhibitor (HDACi); irradiation; pile-up
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Abdollahi, E.; Taucher-Scholz, G.; Jakob, B. Application of Fluorescence Lifetime Imaging Microscopy of DNA Binding Dyes to Assess Radiation-Induced Chromatin Compaction Changes. Int. J. Mol. Sci. 2018, 19, 2399.

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