Correlative Electron Spectroscopic Imaging (ESI) and Electron Tomography of Chromatin.

Generating three-dimensional element-specific images of chromatin, its surrounding protein, and RNA bodies is not routinely practiced in cell or structural biology, but it is an exceptional and powerful tool for understanding chromatin structure in situ. This electron microscopic technique may be a fruitful avenue for those interested in understanding local chromatin structure, the structure-functional relationship of histone modifications on gene expression, or phase separation and RNA regulation of the genome. Here, we describe an approach for performing correlative light and electron spectroscopic imaging tomography, which yields stunning high-resolution structures of chromatin in situ using elemental mapping. Traditional electron spectroscopic imaging (ESI), as all conventional transmission electron microscopy (TEM) image acquisition methods, is restricted to a single image plane, and consequently, information about the z-dimension is collapsed in the image. To overcome this projection limitation, electron tomography approaches are combined with energy-loss imaging; by acquiring and computationally combining a tilt series of image sets, the overlapping fibers of chromatin regions that appear indistinct in 2D are resolved to reveal their 3D architecture. Further combining this approach with correlative light images of the same physical section, structures which are associated with specific proteins of interest can be located and analyzed. Herein, we describe a detailed method for sample preparation, image acquisition, and data analysis and have attached in the Notes the scripts built in-house for ease of use.