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Microscopy Pioneers


Cryosectioning and Immunolabeling: The Contributions of Kiyoteru Tokuyasu


Gareth Griffi ths , 1 Jan-Willem Slot , 2 and Paul Webster 3 * 1 Dept of Biosciences , University of Oslo , 0316 Oslo , Norway 2 Dept of Cell Biology , University Medical Center Utrecht , Utrecht , T e Netherlands 3 Oak Crest Institute of Science , 132 W Chestnut Ave ., Monrovia , CA


* p.webster@oak-crest.org


Today it is taken for granted that ultrathin sections of frozen biological material can be cut, labeled, and observed in a transmission electron microscope (TEM), but to do this successfully for the first time was a totally different story. Below is a short description of how Kiyoteru Tokuyasu (1925–2015) working in San Diego in the 1970s developed the technique for preparing cryosections of fixed and cryoprotected biological material.


By 1973, the conventional method


for ultrastructural analysis was well established. Glutaraldehyde- crosslinked biological material could be contrasted with osmium tetroxide, dehydrated, and embedded in resins for thin sectioning. T is approach, while still widely used for ultrastructure today, is chemically too harsh for preserving antigens if they are to be detected on the sections with specifi c antibodies. Cutting sections of frozen material was already theoretically attractive by the 1960s as a gentler alternative to resins. In fact, Fernandes-Moran had experimented with sectioning unembedded frozen material as early as 1952 [ 1 ]. He was able to produce sections thin enough for examination in the TEM using a modifi ed histology microtome in a -35°C cold room. Poor morphology, together with the introduction of methacrylate resins, led to cryosectioning being temporarily abandoned. It was Tokuyasu who found practical solutions for several key steps of the method that now bears his name [ 2 ] and has made the technique of using thawed cryosectioning such a key technique for cell biology for the last 40 years. T ere were, and still are, two main reasons to consider cutting thin sections of frozen cells or tissues: fi rst to analyze their structure, and second to label sections with antibodies and other reagents aſt er thawing the sections. T e former approach was developed by Jacques Dubochet and colleagues and is now known as cryo EM of vitrifi ed sections (CEMOVIS). A key conceptual breakthrough for this method to work was to understand the process of vitrifi cation, the ability to solidify water so fast that ice crystals cannot form [ 3 ]. Dubochet was awarded the Nobel Prize in Chemistry for this work in 2017, a prize shared with Joachim Frank and Richard Henderson.


44


T e CEMOVIS method is justifi ably referred to as “cryo EM” because the sections are observed in the vitreous state. In contrast, transiently freezing a tissue in order to obtain sections that are subsequently thawed for labeling should not be referred to as cryo EM; nevertheless, the principle of vitrifi cation is important also for the latter approach because it turns out that the specimen must be vitreous to allow it to be sectioned in a useful way. Crystalline ice tends to crumble rather than cut smoothly into sections. T e concept of vitrifying water was not known until 1980


Professor Kiyoteru Tokuyasu


[ 3 , 4 ], and even today vitrifi cation of native specimens is a challenging task. What Tokuyasu discovered serendipitously was that by immersing aldehyde-fi xed cells or tissues in a high concentration of sucrose, the material could be frozen in liquid nitrogen in such a way that it was easy to section at temperatures near -100°C in a cryo-ultramicrotome. Although he was not aware of the vitrifi cation concept per se, he could always see that “well-frozen” samples of most specimens were as transparent as glass. It was later shown that the key role of sucrose was indeed to enable vitrifi cation, even of large samples, and with slow cooling [ 5 ]. Raising the sucrose solution to near saturation (2.1–2.3 M) by the Utrecht group was an important step in the later development of the Tokuyasu technique to a routine method, which is still one of the best and most effi cient methods for immunolocalization studies [ 6 ].


Another fortuitous property of sucrose-infused cryo specimens is that they have the necessary plasticity to allow ribbons of adjacent thin sections to be cut, a key factor in allowing the Tokuyasu method to develop into a routine method. Earlier developments, especially by Wilhelm Bernard and colleagues in Paris, came close to success, using, for example, glycerol rather than sucrose, but the quality of the images that Tokuyasu showed in his 1973 paper [ 2 ] far exceeded any of the preceding publications. Another key innovation in this 1973 paper was the dry cutting of sections. T is avoided chemicals such as DMSO to stretch the sections and allowed the pick up of dry sections from the glass knife (the improved preparation of which was another key Tokuyasu invention) [ 7 ]. Using a cryosectioning bowl designed by A. Kent Christensen (Figure 1), fi tted to a Sorvall MT-2 ultramicrotome [ 8 , 9 ], Tokuyasu was able to obtain thin sections on a dry knife, picking them up using a drop of 2.3 M sucrose on an eyelash to fi sh out and stretch them before transfer to EM grids. Later he replaced this method using


doi: 10.1017/S1551929518000676 www.microscopy-today.com • 2018 July


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