The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.
The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes.
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DOI：
10.1038/srep31417
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发表时间：
2016-08-16
影响因子：
4.6
通讯作者：
Robinson I
中科院分区：
文献类型：
--
作者：
Estandarte AK;Botchway S;Lynch C;Yusuf M;Robinson I
当细胞在细胞周期的不同阶段之间过渡时，染色质经历戏剧性的浓缩和去浓缩。染色质在染色体中的组织仍然是结构生物学的关键挑战之一。荧光寿命成像（FLIM），一种利用荧光团的荧光寿命探测其环境变化的技术，被用来研究固定的人类染色体中染色质致密化的变化。用DNA小沟结合剂DAPI标记固定的人中期和间期染色体，然后使用多光子激发测量和成像荧光寿命。中期染色体扩散中的DAPI寿命变化允许沿着染色体长度绘制染色质的差异压实区域。染色体1、9、15、16和Y的异形区由高度浓缩的组成型异染色质组成，其DAPI寿命值比其余染色体的DAPI寿命值在统计学上显著更短。在DAPI寿命的差异的异形区域表明这些地区的结构的差异。间期核的DAPI寿命变化表明间期染色质致密化和染色体区域形成的变化。染色质致密化差异的成功探测表明FLIM在结构和诊断研究中具有巨大的应用潜力。
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.