Application of fluorescence lifetime imaging microscopy of DNA binding dyes to assess radiation-induced chromatin compaction changes.

Elham Abdollahi,Gisela Taucher-Scholz,Burkhard Jakob

doi:10.3390/ijms19082399

Abstract

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.

Highlights

Research in the past decade has revealed that in eukaryotes chromatin can be classified into higher-order structures with different compaction levels that are critical for the regulation of genome functions [1,2]
Even more critical are double strand breaks (DSBs) induced by high linear energy transfer (LET) radiation like alpha particles or heavy ions, as this type of radiation gives rise to more clustered DNA damage compared to low LET irradiation [6,7,8,9]
To measure chromatin compaction states in living cells, Förster resonance energy transfer (FRET) between histones differently tagged with protein chromophores has been mainly used [23,24,28,31]

Summary

Introduction

Research in the past decade has revealed that in eukaryotes chromatin can be classified into higher-order structures with different compaction levels that are critical for the regulation of genome functions [1,2]. Whereas in the frequency domain a phase shift between synchrony of the modulation of the excitation source and the detector is employed, in the time domain either a gated integration technique or time correlated single photon counting (TCSPC) are used to measure the intensity decay In this case most of the conventional instrumentation used for steady state fluorescence microscopy is not appropriate, since FLIM detects the lifetime of a fluorochrome in the range of few nanoseconds [21]. We established a FLIM-based chromatin compaction assay using DNA binding dyes by which we successfully demonstrated that heterochromatin gets locally decondensed at the sites of ion traversal [29] This finding was indicated by an increase in the lifetime data in nuclei fixed following irradiation. A global chromatin decompaction is shown in living NIH/3T3 cells following X-ray irradiation

Sensitivity of the Organic Dye Sensors upon Chromatin Modulation

Sample Preparation and DNA Staining

Hypertonic Treatment