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Microscopy Pioneers


A modifi ed Sorvall cryo-unit, designed by A. Kent Christensen, similar to the one used by Tokuyasu to obtain his cryosections for labeling. The cryo-chamber was cooled by a jet of nitrogen gas, pumped in by heating liquid nitrogen in a sealed Dewar. When a low temperature was reached, chemically crosslinked tissues cryoprotected in sucrose were frozen onto copper stubs and clamped onto the specimen holder. The holder was suspended in the cryo-chamber and attached to a bridge, which came over the top of the chamber to attach to the specimen arm of the ultramicrotome. A knife was clamped onto the ultramicrotome with a knife clamp fastened to the base of the chamber. The knife could be advanced to the frozen specimen using the knife advance on the front of the chamber. Once the specimen and knife were aligned, sectioning could be performed using the ultramicrotome controls.


a wire loop fi lled with sucrose to pick up the sections from the dry knife—an approach that is still used today. Aſt er picking up the sections, Tokuyasu faced another problem: how to produce contrast in sections while avoiding the serious problem of air-drying before introducing the grids into the vacuum of the electron microscope. His solution was to embed the sections in a relatively thick layer containing heavy metals, such as uranyl acetate, in eff ect (mostly) negatively staining the sections. T e wide range of cells and tissues that he showed as examples in this 1973 paper stand out as excellent examples of ultrastructure [ 2 ], as well as of aesthetics; many examples of EM shown today do not reach this level. However, Tokuyasu then faced another problem. His main goal in developing his technique was to apply it for immuno- localization of antigens, a long passion of his boss, Jon Singer in San Diego. Singer’s group was the fi rst to apply antibodies coupled to ferritin as an electron-dense tag that could be seen by EM, using (poorly preserved) specimens embedded in bovine serum albumin [ 10 ]. T e problem was that ferritin is not very electron opaque. T e much denser colloidal gold was only just being introduced to electron microscopy [ 11 ] with nanoparticle-sized immunogold under 30 nm being visualized in 1975 [ 12 ]. T e result of using a low-density marker was that the denser layer of heavy metal stain, which Tokuyasu needed to support the cryosections during drying, obscured the ferritin label. When the method was applied for the fi rst time in combination with antibody labeling and ferritin [ 13 ],


46


Two fi gures from Tokuyasu’s original 1973 paper in the Journal of Cell Biology [ 2 ]. Tissues were fi xed in 2% glutaraldehyde in 100 mM phosphate buffer for up to 1 hr, infused with 0.6 M sucrose, and frozen onto a copper holder by immersion in liquid nitrogen. Sections were obtained at -70°C using either glass or diamond knives, and the ultramicrotome was cranked by hand. (left) A section of rat skeletal muscle negatively stained with 0.5% aqueous uranyl acetate. Image width approx. 2.5 µm. (right) Part of a plasma cell from mouse spleen. Mitochondria (m) are clearly visible in the negatively stained section. The low contrast reveals endoplasmic reticulum (ER), a multivesicular body (MB), and part of a nucleus (N). The remarkable preservation of mitochondria attracted the attention of microscopists because it was diffi cult to preserve these structures in resin-embedded sections. Image width approx. 2.8 µm.


the results were structurally disappointing since the preser- vation was a compromise between using enough uranyl acetate for contrast and to give some structural preservation, but not enough to obscure the ferritin. T e solution came fi ve years later in a second landmark


paper [ 14 ] in which Tokuyasu described his new trick of embedding the sections on the grids, not with pure uranyl acetate but with a mixture of (a low concentration) of uranyl acetate and 2% methyl cellulose to provide a relatively thick scaff old that dried to a layer thin enough for electrons to easily penetrate. T is allowed both excellent structural preser- vation and clear visualization of ferritin (that was soon to be replaced by colloidal gold). For this embedding step Tokuyasu introduced a wire loop, that has remained an integral part of the Tokuyasu technique, and which is used today in hundreds of labs all over the world. It is also necessary to mention the use of the Tokuyasu method for immunofl uorescence microscopy, an approach fi rst published by Bourguigon et al. [ 15 ]. Over the past 35 years there has been continuous improvement in ultramicrotomes, diamond knives, and in various technical modifi cations used, such as the solutions for section pick-up, etc. T is has turned a demanding, unpredictable


www.microscopy-today.com • 2018 July


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