Large Scale Chromatin Structure

大规模染色质结构是染色质在数百个折叠酶的规模上的高级折叠。对这种折叠知之甚少，但其结构一定会影响DNA的转录、复制和修复。为了更多地了解这些结构，我们以前检查了400 - 2000 kb的转录活性域的光学显微镜。我们发现，几个不同的域在光学显微镜图像中表现出相似的结构，即一系列直径约0.5微米的相邻斑点或“珠”。当这些相同的结构域在转录上无活性时，仅检测到一个点或“珠”。因此，转录诱导从单珠结构到多珠结构的解凝聚。 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。这种成像方式能够以25 nm的分辨率可视化整个未切片的哺乳动物细胞。染色质的检测依赖于有机物质相对于水的吸收所提供的天然对比度。我们的目标是确定染色质结构是否可以通过这种方法检测到，如果可以，则提供25 nm分辨率的核染色质的3D图像。我们目前的研究结果表明，没有足够的对比度来可视化染色质，至少通过透射X射线显微镜。然而，我们已经通过同样的方法获得了细胞质的惊人细节图像。我们现在可以通过分辨率低至30 nm的X射线显微镜清晰地观察细胞中的所有膜结构。这种新方法的优点是，该方法实现了完整的，未染色的3D标本的分辨率，从而提供了前所未有的细胞结构的3D视图。

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.