1. Beam Induced Movement and DDDs Javier Vargas Centro Nacional de Biotecnología-CSIC jvargas@cnb.csic.es

2. The past…

3. Photographic film Directly detects electrons Good signal-to-noise performance (SNR) Slow and laborious (films must be scanned, not possible automation)

4. The past… Photographic film Directly detects electrons Good signal-to-noise performance (SNR) Slow and laborious (films must be scanned, not possible automation) CCD detectors Suitable for automation. Detect photons not electrons, it is needed an scintillator (bad spatial resolution). Bad signal-to-noise performance.

5. The present… Direct-electron Detectors Devices (DDDs) Directly detects electrons. Suitable for automation. Better signal-to-noise ratio than film.

6. The present…

7. The amount of additional noise added by a detector is measured by its detective quantum efficiency (DQE) Ideally a detector would not add any noise and so have a DQE of 1 but all real detectors have values less than 1.

8. The present… Direct-electron Detectors Devices (DDDs) Directly detects electrons. Suitable for automation. Better signal-to-noise ratio than film. Very fast acquisition (17-100s frames/second) We can “see” how the particles behaves as a function of the dose!

9. The present… Direct-electron Detectors Devices (DDDs) Directly detects electrons. Suitable for automation. Better signal-to-noise ratio than film. Very fast acquisition (17-100s frames/second) We can “see” how the particles behaves as a function of the dose! The particles move !!! Beam Induced Movement

10. The present…

12. Brilot, A.F., Chen, J.Z., Cheng, A., Pan, J., Harrison, S.C., Potter, C.S., Carragher, B., Henderson, R., and Grigorieff, N. (2012). Beam-induced motion of vitrified specimen on holey carbon film. J Struct Biol 177, 630-637.

13. The present… Global correction [Li 2013] By frame correction [Bai 2013] Line track correction [Scheres 2014]

14. Li et al., 2013 The alignment method consists in a pure drift correction approach with some local correction dividing each sub-frame area into 3 × 3 regions, and determining the local motion of each region. Fast, converts each movie to an aligned micrograph: Simple connection with standard image processing workflows Not the best option for pronounced local BIM movements [Li 2013]

15. Bai et al., 2013 16 Frame average 8 Frame average 6 Frame average 4 Frame average 2 Frame average

16. Bai et al., 2013

17. Scheres, 2014

18. Accurate in correcting for local and global in- plane BIM movements Optical Flow Fast and “standard” (converts each movie to an aligned micrograph) New Graphical Tool to characterize BIM pattern Abrishami et al., JSB, 2015