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Anchi Cheng, 08/16/2016 06:42 PM
Krios-K2 Presets used for 2.8 A proteasome reconstruction in 5 hours data collection at SEMC @ NYSBC¶
The speed gain relative to previous stage shift data collection is from
- removing iterative movement at Exposure node
- removing queue processing at Exposure node since both need target adjustment and considerable wait time before stable imaging.
- using counting mode. not super-resolution mode on K2.
- Nanoprobe was used to allow smaller beam to be used for multiple targeting on 2 um hole.
Tecnai Krios 300 kV high tension, XFEG, Gun Lens 4 extraction voltage 4100, 70 um C2 aperture and 100 um objective aperture (except when obtaining grid atlas).
Gatan camera dimension 3710(w)x 3838(h) after rotation to Leginon standard. The size of the beam on the scope main viewing screen always covers the 1.2 um hole at en preset on our scope.
Pixel size is 1.10 A at 22500x scope nominal mag.:
| Probe | Lens Series | Magnification: | Preset name: | Image Shift (x,y): | Dimension: | Binning: | Camera Dose (e/camera pixel/s): | Exposure Time (ms): | Specimen Dose Rate (e/A^2): | Spot Size: | Defocus (m): | C2 (um): | frame saving: | Camera |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| micro | LM | 100 | gr | Aligned | 1024x1024 | 4 | n/a | 500 | n/a | 5 | 0.0 | 70 | no | Ceta |
| micro | SA | 400 | sq | Aligned | 927x959 | 4 | 8 | 100 | n/a | 10 | -2e-4 | 70 | no | K2 |
| nano | SA | 1300 | hl | Aligned | 927x959 | 4 | 8 | 200 | 0.004 | 11 | -1.5e-4 | 70 | no | K2 |
| nano | SA | 22500 | fc | 0,0 | 924x924 | 2 | >=8* | 500 | 3.3 | 8 | -7e-7 | 70 | no | K2 |
| nano | SA | 22500 | fa | 0,0 | 927x959 | 4 | >=8* | 250 | 1.65 | 8 | -1.5e-6 | 70 | no | K2 |
| nano | SA | 22500 | en | 0,0 | 3710x3838 | 1 | 8 e/pixel/s and ~0.7 um on the specimen | 7000 | 46 | 8 | (-1e-6 to -2e-6) | 70 | 200 ms/frame |
- Note: If the correlation or power spectrum don't work well enough for the standard Camera Dose Rate of 10 e/pixel/s for fc and fa, you may increase it more so not to have to use longer exposure time. The recommended value in literature is meant to give highest DQE at all sampling frequencies. Using a higher camera dose by 50% will certainly not damage the sensor.
- Note: no defocal pair was acquired in this experiment. Therefore, no ef preset.
Updated by Anchi Cheng over 8 years ago · 1 revisions