Sub-3Å proteasome image-shift data collection » History » Version 2
Anchi Cheng, 08/16/2016 07:10 PM
| 1 | 1 | Anchi Cheng | h1. Sub-3Å proteasome image-shift data collection |
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| 3 | 2 | Anchi Cheng | This data collection obtained same quality data set as stage shift at 2.5x speed with the same presets. Even more speed gain relative to super-resolution mode data collection. |
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| 5 | The speed gain relative to previous stage shift data collection is from |
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| 7 | # removing iterative movement at Exposure node |
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| 8 | # removing queue processing at Exposure node since both need target adjustment and considerable wait time before stable imaging. |
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| 9 | # using counting mode. not super-resolution mode on K2. |
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| 11 | *VERY IMPORTANT*: Measure the amount of beam tilt associated with the image-shift at your scope so that it is not compromising the data. Our scope is at 0.3 mrad at the 1.7 um image shift we applied in this experiment. |
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| 13 | The Transfer Function will reduce all structure factor to 78% at these beamtilts assuming 0.167 mrad per 1 um image shift: |
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| 15 | |image shift (um) |45 degree phase modification resolution(0.78 transfer function amplitude) (Angstroms)| |
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| 16 | |1|2.4| |
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| 17 | |2|3.0| |
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| 18 | |3|3.5| |
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| 19 | |4|3.8| |
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| 21 | 1 | Anchi Cheng | h2. Instruments |
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| 23 | SEMC@NYSBC Krios |
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| 24 | # Scope: FEI Titan-Krios, 300 kV, micro probe at low mag, nano probe at high mags |
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| 25 | # Camera: Gatan K2. mount below Falcon/Ceta. 1.21 Å at 29kx scope nominal mag |
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| 27 | h2. Grid |
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| 29 | * 1.2/1.3 C-flat carbon support; 400 mesh Copper grid. |
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| 31 | [[Krios1-K2-NYSBC]] |
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| 33 | h2. Application: |
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| 35 | MSI-T2 Advanced (= MSI-T + "Beam Tilt Image" for coma-free alignment + "N2 Filling" for Krios auto nitrogen filler) |
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| 37 | h2. Presets Manager: |
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| 39 | No preset cycling (Krios has its own normalization). |
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| 41 | h2. Queuing usage: |
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| 43 | Hole Targeting: Queuing, target at the carbon area to cover four holes. |
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| 44 | Exposure Targeting: *No Queuing*. automated targeting. See [[Exposure Targeting Set-up for MSi-T for Four Holes]] |
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| 46 | h2. Move method to reach the targets: |
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| 48 | Hole: [[Iterative_Stage_Movement|Navigator Iterative movement]]: Target Tolerance: 1e-7 m; Acceptable Tolerance: 3e-7 m |
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| 49 | Focus: Presets Manager image shift |
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| 50 | Exposure: Presets Manager image shift |
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| 52 | h2. Wait time before final exposure: |
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| 53 | 3 seconds |
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| 54 | 10 seconds addition for the first target |