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萧侯
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1 High-resolution imaging from specimens to instruments Xiaojun Huang

3 high-resolution imaging good sample Well prepared cryo sample well aligned TEM with DDD camera Suitable imaging or data collection settings

4 3D reconstruction Frank J. Electron tomography.. methods for three-dimensional visualization of structures in the cell (2ed., Springer, 2006)

5 good sample We can first check the sample with negative staining

6 check the sample with negative staining

7 Well prepared cryo sample

8 Particle in hole prepare cryo sample Continues thin carbon Try different hydrophilic treatment to the grid Use different batch of TEM grid >600kDa try continues thin carbon the air-water interface lower concentration

9 prepare cryo sample Particle overlap lower sample concentration 20nm

10 prepare cryo sample Particle overlap Modify buffer condition Modify purification process 20nm

11 prepare cryo sample Next modify ice thickness 20nm

12 prepare cryo sample

13 prepare cryo sample

14 prepare cryo sample

15 prepare cryo sample Others may help cryo sample preparation Grafix (Chemical Fixation + Gradient Ultracentrifugation) the glycerol, salt, detergent, sugar concentration in the buffer Add different size of protein or virus in the sample

17 well aligned TEM

18 TEM alignment before TEM alignment Adjust Z height Focus Retract objective aperture Condenser aperture centering Condenser astigmation correction Objective astigmation correction Rotation center & coma-free alignment Objective astigmation correction After TEM alignment Camera dark & gain correction

19 coma-free alignment

20 Objective astigmation correction Before correction After correction

21 Suitable imaging or data collection settings Falcon3EC prototype: Normal mode, 47000x, pixel size 1.76A, dose rate 17 e/a2/sec, exp time 2-3 sec, total dose e/a2, defocus -1~-3um Normal mode, 59000x, pixel size 1.42A, dose rate 24 e/a2/sec, exp time sec, total dose e/a2, defocus -1~-3um Counting mode, 75000x, pixel size 1.1A, dose rate 0.83 e/a2/sec, exp time 50 sec, total dose 41.5 e/a2, defocus -1~-3um Counting mode, 96000x, pixel size 0.86A, dose rate 1.35 e/a2/sec, exp time sec, total dose e/a2, defocus -1~-3um K2 summit without GIF: Super resolution mode, 18000x, pixel size 1.35A, dose rate 8e/pix/sec (~4 e/a2/sec), exp time sec, total dose e/a2, defocus -1~- 3um Super resolution mode, 22500x, pixel size 1.04A, dose rate 5e/pix/sec (~4.5 e/a2/sec), exp time 9-13 sec, total dose e/a2, defocus - 1~-3um

22 关键概念电子剂量 (e/å2 vs e/pixel) 总剂量 ( 单位 :e/å2) 对于样品很关键 Tomography 数据收集, 总剂量控制在 e/å2 单颗粒数据收集, 使用可输出每秒多帧的直接电子探测相机, 总剂量蛋白样品常用 40-60e/Å2, 病毒颗粒常用 20-30e/Å2 单颗粒数据收集, 使用不可输出每秒多帧的相机, 总剂量 <30e/Å2 每秒剂量 ( 单位 :e/å2/sec) 对样品稳定性很关键通常 <30 e/å2/sec, 过高会引起样品漂移每秒剂量 ( 单位 :e/pixel/sec) 对直接电子探测相机很关键 Falcon 相机 :50-55 e/pixel/sec K2 相机 super resolution mode:18000x 时用 8e/pixel/sec,22500x 时用 5e/pixel/sec,29000x 及以上可使用 2.5-3e/pixel/sec 总剂量 = 曝光时间每秒剂量每秒剂量 ( 单位 :e/å2/sec) 像素尺寸 ( 单位 :Å) 像素尺寸 ( 单位 :Å) = 每秒剂量 ( 单位 :e/pixel/sec)

23 关键概念像素尺寸相机物理像素尺寸 ( 对数据处理没用 ) Falcon 相机 :14um Gatan 895 CCD:15um 图像像素尺寸 ( 对数据处理非常关键 ) = 相机物理像素大小 / 实际放大倍数电镜控制界面显示的放大倍数不是图像的实际放大倍数, 而是荧光屏上图像的放大倍数最准确的方法是通过测量标样的图像尺寸, 计算得出

24 Nyquist frequency Half of the sampling rate (pixel size) 15 μm Mag 81000X sample Pixel size? detector

25 Nyquist frequency Half of the sampling rate (pixel size) 15 μm Mag 81000X sample Pixel size? detector

26 怎样选取合适的放大倍数 ( 像素尺寸 ) 每个样品颗粒要占据足够数量的像素每张照片至少要涵盖 20 个样品颗粒如果是病毒样品, 每张照片至少要有 3-4 个颗粒像素尺寸小于希望达到的分辨率的两倍

27 Suitable imaging or data collection settings Falcon3EC prototype: Normal mode, 47000x, pixel size 1.76A, dose rate 17 e/a2/sec, exp time 2-3 sec, total dose e/a2, defocus -1~-3um Normal mode, 59000x, pixel size 1.42A, dose rate 24 e/a2/sec, exp time sec, total dose e/a2, defocus -1~-3um Counting mode, 75000x, pixel size 1.1A, dose rate 0.83 e/a2/sec, exp time 50 sec, total dose 41.5 e/a2, defocus -1~-3um Counting mode, 96000x, pixel size 0.86A, dose rate 1.35 e/a2/sec, exp time sec, total dose e/a2, defocus -1~-3um K2 summit without GIF: Super resolution mode, 18000x, pixel size 1.35A, dose rate 8e/pix/sec (~4 e/a2/sec), exp time sec, total dose e/a2, defocus -1~- 3um Super resolution mode, 22500x, pixel size 1.04A, dose rate 5e/pix/sec (~4.5 e/a2/sec), exp time 9-13 sec, total dose e/a2, defocus - 1~-3um

28 Suitable imaging or data collection settings 低倍 ( 低剂量 <0.1e/Å2/sec) 移动至拍照位点高倍拍照

29 Suitable imaging or data collection Low dose imaging Control defocus range monitor dose change settings Adjust coma-free & obj astigmation 1~2 per day

30 12-Jan 15-Jan 25-Jan 16-Feb 23-Feb 29-Feb 17-Mar 23-Mar 26-Mar 29-Mar 03-Apr 08-Apr 17-Apr 20-Apr 24-Apr 28-Apr 01-May 09-May 17-May 22-May 30-May 05-Jun 15-Jun 21-Jun 26-Jun 01-Jul 08-Jul 13-Jul 19-Jul 29-Jul 02-Aug 05-Aug 10-Aug 17-Aug 29-Aug dose rate (e/a2/sec) Titan Krios dose New period: 19-Jun ~ 09-Aug, 45 day. Electron dose increased 16 e/å 2 in two hours. The peak dose was 66 e/å 2.

31 22:12 23:23 0:24 1:23 2:26 3:24 4:27 5:32 6:38 7:51 8:56 10:46 11:59 13:00 14:06 17:56 19:02 20:09 21:14 22:26 23:41 0:43 1:49 2:57 4:07 5:09 6:19 12:51 13:55 14:59 16:16 17:22 18:35 19:47 20:51 21:57 23:17 0:27 1:36 2:47 3:51 6:29 7:37 8:41 Titan Krios dose rate fluctuation 80 total dose(e/a2/sec) on Nov 19-22,

32 high-resolution imaging good sample Well prepared cryo sample well aligned TEM with DDD camera Suitable imaging or data collection settings

35 谢谢!

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