冷冻聚焦离子束-扫描电镜成像技术研究进展

doi:10.11983/CBB21161

植物学报 ›› 2022, Vol. 57 ›› Issue (1): 24-29.DOI: 10.11983/CBB21161

冷冻聚焦离子束-扫描电镜成像技术研究进展

贾星, 孙飞, 季刚()

中国科学院生物物理研究所, 北京 100101

收稿日期:2021-09-14 接受日期:2021-11-17 出版日期:2022-01-01 发布日期:2022-01-17
通讯作者: 季刚
作者简介:* E-mail: jigang@ibp.ac.cn
基金资助:
国家自然科学基金(31801201)

Advances in Cryo-focused Ion Beam-Scanning Electron Microscopy Imaging Technology

Xing Jia, Fei Sun, Gang Ji()

Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

Received:2021-09-14 Accepted:2021-11-17 Online:2022-01-01 Published:2022-01-17
Contact: Gang Ji

摘要/Abstract

摘要： 冷冻聚焦离子束-扫描电镜成像(Cryo-FIB-SEM)是一种新兴的成像检测技术, 在原位进行冷冻聚焦离子束切割和冷冻扫描电镜成像, 为研究天然含水状态下生物样品内部未被破坏的原始结构打开了一扇窗口。近年来, 该技术在生命科学领域的应用研究取得了一系列重要进展。该文对其在冷冻体积连续成像、冷冻光电关联成像、冷冻透射扫描成像、冷冻含水切片制备监控及冷冻扫描图像处理等方面的研究进展进行综述, 并展望了该技术在大体积生物样品三维原位成像研究领域的前沿性发展趋势, 以期推动Cryo-FIB-SEM技术在生物样品三维结构研究中的应用。

关键词: 冷冻聚焦离子束-扫描电镜成像, 冷冻光电关联, 冷冻体积连续成像, 冷冻透射扫描成像

Abstract: Cryo-focused ion beam-scanning electron microscopy (Cryo-FIB-SEM) is an emerging technology designed for advanced imaging detection, which performs in situ by combining Cryo-FIB milling and Cryo-SEM imaging, and has facilitated the visualization of the native structures of biological sample in the context of the cellular environment in the frozen hydrated state. In recent years, a series of important advances have been achieved in the application of this technology in the research field of life science. In this review, we summarize its application in cryo-volume serial imaging, and in combination with cryo-correlative light and electron microscopy (CLEM), cryo-transmission SEM (TSEM), cryo-lamella preparation monitoring, and Cryo-SEM image processing. We also provide future prospective on future development and application of Cryo-FIB-SEM in three-dimensional in situ imaging of large volume biological samples.

Key words: cryo-focused ion beam-scanning electron microscopy (Cryo-FIB-SEM), cryo-correlative light and electron microscopy (Cryo-CLEM), cryo-volume serial imaging, cryo-transmission scanning electron microscopy imaging

贾星, 孙飞, 季刚. 冷冻聚焦离子束-扫描电镜成像技术研究进展. 植物学报, 2022, 57(1): 24-29.

Xing Jia, Fei Sun, Gang Ji. Advances in Cryo-focused Ion Beam-Scanning Electron Microscopy Imaging Technology. Chinese Bulletin of Botany, 2022, 57(1): 24-29.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21161

https://www.chinbullbotany.com/CN/Y2022/V57/I1/24

图/表 3

图1 Cryo-FIB-SEM应用领域 Cryo-FIB-SEM应用领域包括冷冻体积连续成像、冷冻光电关联成像、冷冻透射扫描成像、冷冻含水切片制备监控以及冷冻扫描图像处理。

Figure 1 Cryo-FIB-SEM application fields Cryo-FIB-SEM application fields contain cryo-volume serial imaging, cryo-correlative light and electron microscopy imaging, cryo-transmission scanning electron microscopy imaging, cryo-lamella preparation monitoring, and cryo-SEM image processing.

表1 Cryo-FIB-SEM实验参数总结

Table 1 Summary of serial Cryo-FIB-SEM parameters

	FIB束流(pA)	SEM束流(pA)	SEM电压(kV)	剂量 pA×nS/Å²	驻留时间(ns)	平均线数	像素尺寸x-y-z (nm)	图像尺寸x-y (像素)	切片数	总体积 (μm³)	耗时 (h)
对照	700	59	1.9	22.4	200	19	10×10×20	3072×1150	575	4062	5.5
患者	700	35	2.3	21.3	100	61	10.5×10.5×21	4096×3072	2018	58789	17.5

图2 低温条件下通过不同成像平台进行关联成像的实验流程

Figure 2 Experimental workflow including multiple correlative imaging steps under cryogenic conditions via different imaging platforms

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冷冻聚焦离子束-扫描电镜成像技术研究进展

Advances in Cryo-focused Ion Beam-Scanning Electron Microscopy Imaging Technology

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