三维重构
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两种旋转方式： D为切片内颗粒状结构的病毒、核糖体等 的直径： αn+1= αn+ α0 D为切片厚度，当切片旋转时，电子束穿 透的样品厚度随旋转角度变化： αn+1= αn+ arcsin(sin(α0)cos(αn))
Electron tomography , Joachim Frank
电子断层扫描技术：数据采集条件的确定
分辨率和旋转角度的关系 电子在不同转角照片中的分布 总电子计量 放大倍数(Niquest采样定律) 欠焦量
电子断层扫描技术：数据采集条件的确定
分辨率和旋转角度的关系
如果样品的大小是D( 直径)傅里叶空间取 样率需要高于1/D。 那么为了达到实空间 d的分辨率，在傅里叶空间的半圆(周长 为π/d)中的采样数为(π/d)/(1/D)= πD/d。 也就是旋转步长为180d/πD。
分辨率
• • • • • Signal/noise ratio Masking Determined by two independent map Local resolution Sharpening
Model building （原子模型搭建）：
电子断层扫描三维重构技术
电子断层扫描样品制备
细胞器、细胞等 大至数百微米
冷冻替换，树脂包埋 (freeze substitution, resin embedding) 常温切片 (room temperature sectioning)
电子断层扫描样品制备：高压冷冻
样品大小、降温速率和冰相的关系
高压冷冻仪
高压能阻碍晶体冰的形成，减少形成晶体冰时的放热，增快样品降温速率
Particle boxing (颗粒挑选）
1. cross correlation
由于
颗粒挑选
二维分类
三维分类
Refinement（结构优化）:
1 Initial model building (random angle, masked noise, tomogram, negative stain, tilt pair…) 2 Orientation determination (projection match, maximum likelihood, real space, reciprocal space) 3 Reconstruction (real space, reciprocal space…) 4 Iteration
电子在不同转角照片中的分布
电子断层扫描技术：数据采集条件的确定
总电子计量
电子晶体学 0.5~4e/Å 2 单颗粒方法 10~50e/Å 2
电子断层扫描 100~250e/Å 2
旋转步长1.5度，总采集图像93，每张图像的电子数约为2e/Å2，总电子计数为 ~200e/Å2
Taylor and Glaeser (1976) J. Ultrastruc. Res. 55:448
Dose based weighting
Ne(k) is the critical exposure at which the intensity of the diffraction spot fades to 1/e times its initial intensity
CTF fitting 衬度传递函数参数确定(ctf per particle, local ctf, ctf per microgragh) Fimage(s) = F(s) CTF(s) Env(s) + Noise(s) Power spectrum
Electron tomography , p139， Joachim Frank
电子断层扫描技术：硬件选择
相位板应用
Journal ofMicroscopy, Vol. 259, Issue 1 2015, pp. 74–78
电子断层扫描样品制备：冷冻切片
Compression, crevasses and knife marks
切片厚度： A-H分别是 10，20， 40，60， 80，100， 150，200 纳米。
Electron tomography p62, Joachim Frank
Han et al., Journal of Microscopy, Vol. 230, Pt 2 2008, pp. 167 –171
Electron tomography , p 130, Joachim Frank
电子断层扫描技术：硬件选择
能量过滤系统 相位板成像 直接电子探头的应用
电子断层扫描技术：硬件选择 能量过滤系统
FIGURE 10. Filtered and unfiltered images of ice-embedded specimens. (a) Actin filaments and vesicles (250 nm thick ice). The half of the image below the diagonal is zero-loss filtered, while the upper half is unfiltered. Whereas contrast is comparable in both images, the resolution is improved in the case of the filtered image, such that individual actin filaments can be identified. (b) Multilamellar vesicles (600 nm thick ice). The division of the image is as in (a). Only a few lamella can be distinguished in the unfiltered case. (From Koster et al. (1997), reproduced with permission of Elsevier).
数据采集
Radiation Damage 辐照损伤 • Electron beam interacts with sample and inelastic scattering damages sample • Damage is proportional to total dose (e/Å2), not doserate dependent • Damage is significant for organic material but less severe for inorganic material • Depends on resolution, maximal dose is in the range of (1-50e/Å2) • Radiation damage limits usefulness of TEM data regardless of initial quality of specimen • Radiation damage instead of instrument resolving power is the limiting factor to reach atomic resolution
Micron 42 (2011) 117–131
• 负染 • 快速冷冻
Vitrobot
EMGP
样品大小、降温速率和冰相的关系
优势取向问题
Nat Commun. Байду номын сангаас015; 6: 7548.
蛋白质解体问题
Protein Sci. 2016 Sep 26. doi: 10.1002/pro.3048.
电子断层扫描样品制备：FIB减薄
电子断层扫描样品制备：FIB减薄
Ion beam
无假象，产量低
Villa et al., Current Opinion in Structural Biology Volume 23, Issue 5, October 2013, Pages 771–777
电子断层扫描技术：数据采集条件的确定
J. Ultrastruc. Res. 55:448 1976
Low Dose Imaging 低剂量成像
• Minimum current density needed to barely see an object on fluorescent screen is ~1 e/μm2 /sec at the screen -> dose rate at specimen: 4 e/Å2/ sec at 20,000X • Hence, not enough time to visualize let alone focus and record images of biological specimens before they are damaged • Low-dose imaging is essential to minimize radiation in the usable sample region
Electron tomography p19, Joachim Frank
高频信息的破坏和样品沿z轴方向的变形
单颗粒分析方法
由于不同颗粒在三维空间具有全同性， 可以对大量颗粒的成像数据求平均，提 信噪比。
电子断层成像三维重构
不同角度的数据全部来自同一个生物样 品，总电子计量上限非常低，导致三维 重构信噪比差，从而限制了分辨率 （4-10nm）。
负染 (negative stain) 常规冷冻 (plunge freezing)
蛋白质复合物、病毒等 几十到上百纳米
高压冷冻 (high pressure freezing) 冷冻切片 (cryo sectioning) 离子束减薄 (focused ion beam milling)
电子断层扫描技术：数据采集条件的确定
放大倍数(Niquest采样定律)以及欠焦量
FIGURE 7. The effect of the defocus on the appearance of images for cryotomography. Images of ice-embedded particles (tricorn capsid of Thermoplasma acidophilum) at 0.5 μm (left) and 5 μm (right) underfocus. The spherical shaped particles are clearly visible at large defocus. (From Walz et al. (1997a), reproduced with permission of Cell Press).
Automatic data collection! ~1000 images per day.
数据处理
Motion correction (micrograph based VS particle based)
Structure. 2012 Nov 7; 20(11): 1823–1828.
Nat Methods. 2013 Jun;10(6):584-90.
冷冻电镜三维重构原理和过程简介
中心截面原理
单颗粒技术
单颗粒法三维重构的流程
制样
• 负染 • 冷冻
J. Vis. Exp. (58), e3227 10.3791/3227
Anionic: PTA , methylamine tungstate , silicon tungstate… Cationic: uranyl acetate (UA), UF, uranyl nitrate…
钼酸铵 Tomato bushy stunt virus 番笳丛矮病毒 (modified from Harris and Scheffler, 2002)
Octopus hemocyanin decamers, 章鱼血蓝蛋白十聚体。 The scale bars indicate 100 nm.
Electron tomography , Joachim Frank
电子断层扫描技术：数据采集条件的确定
分辨率和旋转角度的关系 电子在不同转角照片中的分布 总电子计量 放大倍数(Niquest采样定律) 欠焦量
电子断层扫描技术：数据采集条件的确定
分辨率和旋转角度的关系
如果样品的大小是D( 直径)傅里叶空间取 样率需要高于1/D。 那么为了达到实空间 d的分辨率，在傅里叶空间的半圆(周长 为π/d)中的采样数为(π/d)/(1/D)= πD/d。 也就是旋转步长为180d/πD。
分辨率
• • • • • Signal/noise ratio Masking Determined by two independent map Local resolution Sharpening
Model building （原子模型搭建）：
电子断层扫描三维重构技术
电子断层扫描样品制备
细胞器、细胞等 大至数百微米
冷冻替换，树脂包埋 (freeze substitution, resin embedding) 常温切片 (room temperature sectioning)
电子断层扫描样品制备：高压冷冻
样品大小、降温速率和冰相的关系
高压冷冻仪
高压能阻碍晶体冰的形成，减少形成晶体冰时的放热，增快样品降温速率
Particle boxing (颗粒挑选）
1. cross correlation
由于
颗粒挑选
二维分类
三维分类
Refinement（结构优化）:
1 Initial model building (random angle, masked noise, tomogram, negative stain, tilt pair…) 2 Orientation determination (projection match, maximum likelihood, real space, reciprocal space) 3 Reconstruction (real space, reciprocal space…) 4 Iteration
电子在不同转角照片中的分布
电子断层扫描技术：数据采集条件的确定
总电子计量
电子晶体学 0.5~4e/Å 2 单颗粒方法 10~50e/Å 2
电子断层扫描 100~250e/Å 2
旋转步长1.5度，总采集图像93，每张图像的电子数约为2e/Å2，总电子计数为 ~200e/Å2
Taylor and Glaeser (1976) J. Ultrastruc. Res. 55:448
Dose based weighting
Ne(k) is the critical exposure at which the intensity of the diffraction spot fades to 1/e times its initial intensity
CTF fitting 衬度传递函数参数确定(ctf per particle, local ctf, ctf per microgragh) Fimage(s) = F(s) CTF(s) Env(s) + Noise(s) Power spectrum
Electron tomography , p139， Joachim Frank
电子断层扫描技术：硬件选择
相位板应用
Journal ofMicroscopy, Vol. 259, Issue 1 2015, pp. 74–78
电子断层扫描样品制备：冷冻切片
Compression, crevasses and knife marks
切片厚度： A-H分别是 10，20， 40，60， 80，100， 150，200 纳米。
Electron tomography p62, Joachim Frank
Han et al., Journal of Microscopy, Vol. 230, Pt 2 2008, pp. 167 –171
Electron tomography , p 130, Joachim Frank
电子断层扫描技术：硬件选择
能量过滤系统 相位板成像 直接电子探头的应用
电子断层扫描技术：硬件选择 能量过滤系统
FIGURE 10. Filtered and unfiltered images of ice-embedded specimens. (a) Actin filaments and vesicles (250 nm thick ice). The half of the image below the diagonal is zero-loss filtered, while the upper half is unfiltered. Whereas contrast is comparable in both images, the resolution is improved in the case of the filtered image, such that individual actin filaments can be identified. (b) Multilamellar vesicles (600 nm thick ice). The division of the image is as in (a). Only a few lamella can be distinguished in the unfiltered case. (From Koster et al. (1997), reproduced with permission of Elsevier).
数据采集
Radiation Damage 辐照损伤 • Electron beam interacts with sample and inelastic scattering damages sample • Damage is proportional to total dose (e/Å2), not doserate dependent • Damage is significant for organic material but less severe for inorganic material • Depends on resolution, maximal dose is in the range of (1-50e/Å2) • Radiation damage limits usefulness of TEM data regardless of initial quality of specimen • Radiation damage instead of instrument resolving power is the limiting factor to reach atomic resolution
Micron 42 (2011) 117–131
• 负染 • 快速冷冻
Vitrobot
EMGP
样品大小、降温速率和冰相的关系
优势取向问题
Nat Commun. Байду номын сангаас015; 6: 7548.
蛋白质解体问题
Protein Sci. 2016 Sep 26. doi: 10.1002/pro.3048.
电子断层扫描样品制备：FIB减薄
电子断层扫描样品制备：FIB减薄
Ion beam
无假象，产量低
Villa et al., Current Opinion in Structural Biology Volume 23, Issue 5, October 2013, Pages 771–777
电子断层扫描技术：数据采集条件的确定
J. Ultrastruc. Res. 55:448 1976
Low Dose Imaging 低剂量成像
• Minimum current density needed to barely see an object on fluorescent screen is ~1 e/μm2 /sec at the screen -> dose rate at specimen: 4 e/Å2/ sec at 20,000X • Hence, not enough time to visualize let alone focus and record images of biological specimens before they are damaged • Low-dose imaging is essential to minimize radiation in the usable sample region
Electron tomography p19, Joachim Frank
高频信息的破坏和样品沿z轴方向的变形
单颗粒分析方法
由于不同颗粒在三维空间具有全同性， 可以对大量颗粒的成像数据求平均，提 信噪比。
电子断层成像三维重构
不同角度的数据全部来自同一个生物样 品，总电子计量上限非常低，导致三维 重构信噪比差，从而限制了分辨率 （4-10nm）。
负染 (negative stain) 常规冷冻 (plunge freezing)
蛋白质复合物、病毒等 几十到上百纳米
高压冷冻 (high pressure freezing) 冷冻切片 (cryo sectioning) 离子束减薄 (focused ion beam milling)
电子断层扫描技术：数据采集条件的确定
放大倍数(Niquest采样定律)以及欠焦量
FIGURE 7. The effect of the defocus on the appearance of images for cryotomography. Images of ice-embedded particles (tricorn capsid of Thermoplasma acidophilum) at 0.5 μm (left) and 5 μm (right) underfocus. The spherical shaped particles are clearly visible at large defocus. (From Walz et al. (1997a), reproduced with permission of Cell Press).
Automatic data collection! ~1000 images per day.
数据处理
Motion correction (micrograph based VS particle based)
Structure. 2012 Nov 7; 20(11): 1823–1828.
Nat Methods. 2013 Jun;10(6):584-90.
冷冻电镜三维重构原理和过程简介
中心截面原理
单颗粒技术
单颗粒法三维重构的流程
制样
• 负染 • 冷冻
J. Vis. Exp. (58), e3227 10.3791/3227
Anionic: PTA , methylamine tungstate , silicon tungstate… Cationic: uranyl acetate (UA), UF, uranyl nitrate…
钼酸铵 Tomato bushy stunt virus 番笳丛矮病毒 (modified from Harris and Scheffler, 2002)
Octopus hemocyanin decamers, 章鱼血蓝蛋白十聚体。 The scale bars indicate 100 nm.