It was in this journal, in 2006, that Mike Marko and coworkers from Albany (US) published a paper in which they demonstrated the possibility of using a focussed ion beam (FIB) to prepare thin sections (i.e. lamellae) from frozen hydrated Life Science samples. The so-called cryo-lamellae were subsequently transferred to a cryotransmission electron microscope (cryo-TEM) to record images of cells embedded in vitreous ice.1 Their work sparked a lot of activities around the globe, with multiple groups developing their own ‘cryo-TEM prep’ methods. A small but steadily growing community of cryo-FIB-users started organising small workshops across Europe (Colerain,UK, 2008;Utrecht, TheNetherlands, 2009; Vlaardingen, The Netherlands, 2012; Lausanne, Switzerland, 2014; Nottingham, UK, 2017). The discussions between the groups clearly helped and inspired the development of various approaches towards cryo-TEM preparation to where we are today. Today’s status can be seen in the literature, as what is being published is not only describing further innovative cryo-TEM preparation methodologies, but also describing scientific contributions to various fields in Life Sciences based on established cryo-TEM preparation methods. Cryo-TEM preparation is becoming a highend standard technique for high-end scientific questions. Hence, this special issue of Journal of Microscopy clearly reflects that cryo-TEM preparation is the main application for cryo-FIB instruments. For this special issue, we have brought back together colleagues from those early days of cryo-TEM preparation developments to both reflect on their techniques and to look ahead of where to go next. Hence, primarily discussed in this special issue are the three routes towards cryo-TEM observations of cryo-FIBmade lamellae: Thinning cells on a grid,2 lift-out techniques2,3 and thinning of high-pressure frozen samples.4 The cryo-TEM preparation application is the result of about three decades of developing cryo-FIB techniques. The FIB milling technique had become popular in semiconductor research in the 1980s. However, it was recognised that the FIB introduces defects and other types of damage in the samples. Meanwhile, preliminary work in the late 1980s resulted in successful observations of frozen hydrated Life Science samples in a cryo-scanning electron microscope (SEM) in the very early 1990s.5 Soon, FIB milling was performed under cryo-conditions to mitigate the damaging effect of the ion beam.6 Around that time, the combined FIB-SEM instruments became increasingly popular. With ongoing improvements of FIB-SEM instruments, the first feasibility studies of cryo-FIB-SEM work on frozen hydrated Life Science samples were published by Hans Mulders7 and Ingo Gestman et al.8 The potential of the technique was quickly recognised and resulted in where we are today. Thus, the work of cryo-TEM preparation is based upon three decades of developing cryo-FIB-SEM techniques. This is even longer if we include various ways of preparing Life Science samples. Therefore, the extensive basics of cryo-FIB operations, including a detailed description of the preparation and transfer options are discussed in the paper by Mike Hayles and Matthijs de Winter.9 Their aim is to inspire and help forward newcomers to the field and to serve as a general resource for more experienced cryoFIB-SEM operators. One could say that cryo-FIB in Life Sciences has matured in less than 20 years, but that does not mean there is nothing left to do. Jakub Kuda and coworkers2 discuss cryo-FIB-SEM tomography of unstained biological samples, a technique first introduced by Andreas Schertel in 2013.10 The FIB is used to mill away thin consecutive slices and the resulting cross sections are imaged by the SEM. The series of SEM images is used for a digital reconstruction of the analysed volume. The results of cryo-FIBSEM tomography are very promising, but the image formation mechanism is not yet fully understood. The image is formed by depositing charge at or just behind the cross section which affects the secondary electron generation of subsequent electron beam scans. Needless to say, the SEM scan strategy requires precise tuning. The kind of cellular features that can be observed and to what detail is still a question to be researched. Another development which has been researched recently is the potential all-in-one instrument,4,11 when not only the making of cryo-TEM lamella is done in the FIB-SEM instrument, but also the observations are made in the FIB-SEM instrument. The latter is done by placing a solid-state detector underneath the cryo-TEM lamella and performing transmission imaging with the SEM. This would be a complimentary technique to, for example, the

中文翻译：

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生命科学（及其他领域）中的低温聚焦离子束

2006 年，来自奥尔巴尼（美国）的 Mike Marko 和同事在这本期刊上发表了一篇论文，他们在论文中展示了使用聚焦离子束 (FIB) 从冷冻水合生命科学中制备薄片（即薄片）的可能性样品。所谓的冷冻薄片随后被转移到冷冻透射电子显微镜 (cryo-TEM) 上，以记录嵌入玻璃冰中的细胞的图像。1 他们的工作在全球范围内引发了许多活动，多个小组开发了自己的“冷冻” -TEM prep' 方法。一个规模虽小但稳步增长的低温 FIB 用户社区开始在欧洲组织小型研讨会（Colerain，英国，2008 年；乌得勒支，荷兰，2009 年；弗拉尔丁根，荷兰，2012 年；瑞士洛桑，2014 年；诺丁汉，英国，2017 年） . 小组之间的讨论显然有助于并激发了各种方法的发展，以将低温 TEM 制备到我们今天的位置。今天的状态可以在文献中看到，因为所发表的不仅描述了进一步创新的低温 TEM 制备方法，而且还描​​述了基于已建立的低温 TEM 制备方法对生命科学各个领域的科学贡献。Cryo-TEM 制备正在成为解决高端科学问题的高端标准技术。因此，本期《Journal of Microscopy》清楚地反映了cryo-TEM 制备是cryo-FIB 仪器的主要应用。对于本期特刊，我们召集了早期冷冻 TEM 制备发展的同事，以反思他们的技术并展望下一步的发展方向。因此，在本期特刊中主要讨论的是对冷冻 FIB 制成的薄片进行冷冻 TEM 观察的三种途径：网格上的细胞变薄、2 提升技术 2、3 和高压冷冻样品的变薄。 4 冷冻 TEM制备应用是大约三个十年发展冷冻 FIB 技术的结果。FIB 铣削技术在 1980 年代已在半导体研究中流行起来。然而，人们认识到 FIB 会在样品中引入缺陷和其他类型的损坏。同时，1980 年代后期的初步工作导致在 1990 年代早期在冷冻扫描电子显微镜 (SEM) 中成功观察到冷冻水合生命科学样品。5 很快，FIB 研磨在冷冻条件下进行，以减轻冷冻水合生命科学样品的破坏性影响。离子束。6 大约在那个时候，FIB-SEM 组合仪器变得越来越流行。随着 FIB-SEM 仪器的不断改进，Hans Mulders7 和 Ingo Gestman 等人发表了关于冷冻水合生命科学样品的第一份 cryo-FIB-SEM 可行性研究。我们今天。因此，低温 TEM 的制备工作基于三个十年发展的低温 FIB-SEM 技术。如果我们包括准备生命科学样品的各种方法，这会更长。所以，Mike Hayles 和 Matthijs de Winter 在论文中讨论了 cryo-FIB 操作的广泛基础知识，包括对制备和转移选项的详细描述。9 他们的目的是激励和帮助新人进入该领域，并作为一个为更有经验的 cryoFIB-SEM 操作员提供一般资源。可以说生命科学领域的冷冻 FIB 在不到 20 年的时间里已经成熟，但这并不意味着没有什么可做的。Jakub Kuda 和同事 2 讨论了未染色生物样品的冷冻-FIB-SEM 断层扫描，这是 Andreas Schertel 在 2013.10 首次引入的技术。FIB 用于磨掉薄的连续切片，并通过 SEM 对生成的横截面进行成像。该系列的 SEM 图像用于分析体积的数字重建。冷冻 FIBSEM 断层扫描的结果非常有希望，但图像形成机制尚未完全了解。图像是通过在影响后续电子束扫描的二次电子生成的横截面处或其后面沉积电荷形成的。不用说，SEM 扫描策略需要精确调整。可以观察到什么样的细胞特征，具体到什么细节仍然是一个有待研究的问题。最近研究的另一项发展是潜在的一体化仪器，4,11 当不仅在 FIB-SEM 仪器中完成冷冻 TEM 薄片的制作，而且在 FIB-SEM 中进行观察时乐器。后者是通过在低温 TEM 薄片下方放置一个固态探测器并使用 SEM 进行透射成像来完成的。