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Reproduced with permissions from (a)Medalia et al. As optics improved over another three centuries, so did our knowledge of cell ultrastructure and function. The development of chromogenic stains enabled the visualization of otherwise invisible intracellular bodies, including chromosomes inside the nucleus. In the late 19thcentury Ernst Abbe acknowledged, however, that this resolution of light microscopes would ultimately be limited to about half the wavelength of Roy-Bz light. The invention of the electron microscope by Ruska in the early 1930s and the availability of commercial instruments shortly thereafter promised a new era of cell biology in which resolution could be extended by at least two orders of magnitude. Despite this transformative technological advance, the first images of subcellular structures such as ribosomes, viruses, and the cytoskeleton were not obtained for approximately two more decades. Some of the principal challenges were that biological specimens are hydrated and radiation sensitive, exhibit poor intrinsic contrast, and are often too thick to be imaged intact. To solve these problems, methods were developed to chemically fix and stain specimens, embed them in plastic resins, and section them with a microtome. Together, these techniques enabled a Golden age of rapid discovery in the 1950s to 1960s (Fig. 1). == Fig. 1. == Timeline of key developments. Key technologies and applications of EM are shown. The gray gradients emphasize that each of these development took many years. EM images are 2-D projections. To gain the perspective of depth, stereoscopy was introduced, in which two images are taken of a specimen one at positive tilt and the other at unfavorable tilt. The pair of images were viewed with a stereoscope, and quantitative data could be generated by measuring relative displacements of objects perpendicular to the tilt axis and then calculating their separation in Z Rabbit Polyclonal to NF1 (the direction parallel to the electron beam) using the known tilt angles. But since stereoscopy uses only two views of the specimen, the resolution in Z is limited. Another way to Roy-Bz obtain 3-D information is usually to cut a sample into a series of thin sections and image each section individually. As each section represents a resolution element in Z, an entire cell can then be reconstructed in 3-D by computationally stacking the images. Reconstructions from serial sections have Roy-Bz an anisotropic resolution of ~1-2 nm in the cutting plane (limited by the stain granularity), but only 20 nm perpendicular to the cutting direction (limited by how thin sections can be cut and handled) (Mastronarde et al., 2000). Fine structural details of cellular organization are often further obfuscated by the losses that occur around the cutting planes, sample shrinkage and warping caused by radiation damage, and the difficulty of tracking features between sections (especially features that lie in a cutting plane). Nevertheless, since it is usually relatively straightforward to do in any laboratory with an EM, this method has been used since the 1950s to map out the ultrastructure of various eukaryotic cells and tissues. As an example of the power of this technique, theC. elegansconnectome the wiring diagram of all 302 neurons in this millimeter-long worm was reconstructed by serial EM (White et al., 1986). A third method to obtain high-resolution 3-D EM images of at least the surface of biological objects is usually scanning electron microscopy. Newer methods that combine sequential focused-ion beam milling with SEM (also called ion-abrasion scanning electron microscopy) are beginning to uncover internal detail as well, as exhibited in a recent.