title: 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.
authors.affiliation: London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK.|Research Complex at Harwell, Rutherford Appleton Laboratory, Oxon, OX11 0FA, UK.
authors.forename: Ana Katrina
authors.initials: AK
authors.lastname: Estandarte
authors.affiliation: Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxon, OX11 0QX, UK.
authors.forename: Stanley
authors.initials: S
authors.lastname: Botchway
authors.affiliation: London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK.|Research Complex at Harwell, Rutherford Appleton Laboratory, Oxon, OX11 0FA, UK.
authors.forename: Christophe
authors.initials: C
authors.lastname: Lynch
authors.affiliation: London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK.|Research Complex at Harwell, Rutherford Appleton Laboratory, Oxon, OX11 0FA, UK.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Yusuf
authors.affiliation: London Centre for Nanotechnology, University College London, London, WC1H 0AH, UK.|Research Complex at Harwell, Rutherford Appleton Laboratory, Oxon, OX11 0FA, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Robinson
mesh_terms: D001402:B-Lymphocytes / Q000502:physiology; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D002877:Chromosomes, Human / Q000378:metabolism*; D004847:Epithelial Cells / Q000502:physiology; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007211:Indoles / Q000032:analysis*; D007399:Interphase; D008677:Metaphase; D061848:Optical Imaging / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D002843:Chromatin; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI