Large Scale Chromatin Structure

大尺度染色质结构是染色质在几百个碱基的尺度上的高阶折叠。人们对这种折叠知之甚少，但这种结构肯定会影响DNA的转录、复制和修复。为了更多地了解这些结构，我们以前用光学显微镜检查了400-2000kb的转录活性结构域。我们发现，在光学显微镜图像中，几个不同的结构域显示出相似的结构，即一系列直径约0.5微米的相邻点状或“珠子”。当这些相同的结构域在转录上不活跃时，只检测到一个标点或“珠子”。因此，转录诱导了从单珠状结构到多珠状结构的解凝聚。为了以更高的分辨率观察这些结构，我们正在探索X射线显微镜的适用性。这种成像方式使完整的、未切开的哺乳动物细胞能够以25纳米的分辨率可视化。染色质的检测依赖于有机物相对于水的吸收所提供的自然对比。我们的目标是确定是否可以用这种方法检测到染色质结构，如果可以，那么提供25 nm分辨率的核染色质的3D图像。我们目前的结果表明，至少在透射式X射线显微镜下，没有足够的对比度来显示染色质。然而，我们已经通过同样的方法获得了细胞质的惊人的详细图像。我们现在可以通过x射线显微镜清楚地观察到细胞中的所有膜结构，分辨率可达30 nm。这种新方法的优点是，该方法实现了对完整的、未染色的3D样本的分辨率，从而提供了前所未有的细胞结构的3D视图。我们目前正在考虑提高染色质结构对比度的方法，包括发展相位对比X射线显微镜或通过标准吸收X射线显微镜对冰冻核切片进行成像。

Large-scale chromatin structure is the higher-order folding of chromatin on the scale of several hundred kilobases. Little is known about this folding, but the structures must influence transcription, replication and repair of DNA. To learn more about these structures, we previously examined transcriptionally active domains of 400 - 2000 kb by light microscopy. We found that several different domains exhibit similar structures in light microscope images, namely a series of adjacent puncta or "beads" approximately 0.5 microns in diameter. When these same domains are transcriptionally inactive, only one punctum or "bead" is detected. Thus transcription induces a decondensation from single beaded to multi-beaded structures. To observe these structures at higher resolution we are exploring the applicability of x-ray microscopy. This imaging modality enables visualization of whole, unsectioned mammalian cells at 25 nm resolution. Detection of chromatin relies on the natural contrast afforded by the absorption of organic matter relative to water. Our goal is to determine if chromatin structure can be detected by this approach and if so, then to provide a 3D image of nuclear chromatin at 25 nm resolution. Our current results suggest that there is not sufficient contrast to visualize chromatin, at least by transmission x-ray microscopy. However, we have obtained strikingly detailed images of the cytoplasm by the same approach. We can now clearly visualize all membrane structures in the cell by x-ray microscopy down to 30 nm resolution. The advantage of this new approach is that the method achieves this resolution for intact, unstained 3D specimens, thereby providing an unprecedented 3D view of cellular architecture. We are presently considering ways to improve contrast of chromatin based structures, including development of phase contrast x-ray microscopy or imaging by standard absorption x-ray microscopy of frozen nuclear sections.