The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes

Abstract

Chromatin undergoes dramatic condensation and decondensation as cells transition between the different phases of the cell cycle. The organization of chromatin in chromosomes is still one of the key challenges in structural biology. Fluorescence lifetime imaging (FLIM), a technique which utilizes a fluorophore’s fluorescence lifetime to probe changes in its environment, was used to investigate variations in chromatin compaction in fixed human chromosomes. Fixed human metaphase and interphase chromosomes were labeled with the DNA minor groove binder, DAPI, followed by measurement and imaging of the fluorescence lifetime using multiphoton excitation. DAPI lifetime variations in metaphase chromosome spreads allowed mapping of the differentially compacted regions of chromatin along the length of the chromosomes. The heteromorphic regions of chromosomes 1, 9, 15, 16, and Y, which consist of highly condensed constitutive heterochromatin, showed statistically significant shorter DAPI lifetime values than the rest of the chromosomes. Differences in the DAPI lifetimes for the heteromorphic regions suggest differences in the structures of these regions. DAPI lifetime variations across interphase nuclei showed variation in chromatin compaction in interphase and the formation of chromosome territories. The successful probing of differences in chromatin compaction suggests that FLIM has enormous potential for application in structural and diagnostic studies.

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

“…This is in line with recent studies which described the application of organic DNA dyes as FLIM sensors for chromatin structure [26,27]. However, the molecular mechanism behind the lifetime-dependency of the single dyes on chromatin compaction is not known.…”

Non-Random Mis-Segregation of Human Chromosomes

“…Alternatively, it is possible that differences in centromeric composition underlie the sensitivity of chromosomes 1 and 2 to cohesion fatigue. Of note, large regions of pericentric heterochromatin have been identified at the q arms of chromosomes 1, 3, 4, 9, 16 and 19 (Atkin and Brito-Babapulle, 1981;Craig-Holmes and Shaw, 1971;Estandarte et al, 2016) (Figure 1a) although it is not clear whether the nature of chromosome 1 pericentric heterochromatin differs qualitatively, and how this might render chromosomes prone to cohesion fatigue and merotelic attachment.…”

The squared distance approach to frequency domain time‐resolved fluorescence analysis

“…Simulation I represents a general case of three widely spaced FLTs. Simulation II represents common closely FLT values, which correspond to FLTs of DAPI bound to DNA of leukocytes in different situations .…”