Full Protocol on a F30 with an energy filter » History » Version 3
Amber Herold, 06/21/2010 12:19 PM
1 | 1 | Amber Herold | h1. Full Protocol on a F30 with an energy filter |
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7 | This is edited from a complete protocol written by Lu Gan (Jensen Lab, Caltech) for users |
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8 | who have not used general MSI applications. It is modified to reflect the current Leginon |
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9 | version and a current and graphic-rich protocol by Jian Shi (contact him at |
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10 | jianshi@caltech.edu for a copy) of the same group. |
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14 | h2. Notes |
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18 | * Make sure EFTEM is always on |
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21 | * If presets manager has error, disable then enable "Filtered" box in |
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22 | Tecnai/TUI/GIF> menu. (TUI: Tecnai User Interface) |
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25 | * Leginon re-aligns ZLP, using a "reference" hole, after each tilt series. |
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28 | * Pause Leginon (for filling dewars, etc.): wait until tilt series is almost done, |
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29 | then press "PAUSE" in FOCUS node. Leginon will pause at start of next tilt |
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30 | series. |
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33 | * Overall scheme: Take grid atlas. At reference position, do cross-over correction, |
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34 | align presets, center objective aperture. Select and preview targets. Prepare data |
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35 | collection (gr and sq presets = skip sequence). Take dose in tomography setting. Let |
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36 | objective lens equilibrate. Align ZLP, insert objective aperture, fire away! |
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44 | h2. Initial setup |
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48 | 2 | Amber Herold | # Tecnai/GIF> Tune GIF with scope column empty |
49 | # Tecnai/TUI/Stage> Set Z = 0. |
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50 | # Tecnai/TUI> Turn on Intensity Zoom |
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51 | # Tecnai> Remove objective aperture. |
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52 | # Quit UCSF Tomo, TEM Auto-functions, Low-Dose kit. |
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60 | h2. Start Leginon |
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62 | 2 | Amber Herold | # Launch Leginon |
63 | # Choose either to start a new session or continue an old one, then launch Multiscale |
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64 | 1 | Amber Herold | Tomography from Applications menu. |
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72 | h2. Presets setup with empty column |
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76 | 2 | Amber Herold | # Leginon/Presets Manager> Set up all the presets except align image shifts. If |
77 | 1 | Amber Herold | starting new session, import presets from previous session. If you want to preview |
78 | images at high mag, make or copy a preview called "preview," which should be set to 3x3 |
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79 | to 4x4 binning for greater contrast. |
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87 | h2. Flat fielding and dose calibration |
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91 | 2 | Amber Herold | # Tecnai/TUI/EFTEM> Make sure that "filter" is on. |
92 | # Leginon/Presets Manager> tomo preset to scope. |
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93 | # Tecnai/TUI/GIF> Go back to hole and re-align ZLP (and tune GIF if it's been ~24 |
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94 | 1 | Amber Herold | hours since last |
95 | 2 | Amber Herold | # Leginon/Correction/Settings> set camera configuration to match that of the |
96 | 1 | Amber Herold | preset. Choose to use 2 correction channels. |
97 | 2 | Amber Herold | # Leginon/Correction> acquire dark current, then acquire bright image, then |
98 | 1 | Amber Herold | acquire corrected image to make sure "flat-fielding" worked. |
99 | 2 | Amber Herold | # Leginon/Presets manager> redo dose measurement. |
100 | # Leginon/Tomography> recheck dose. |
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101 | # Leginon/Correction> repeat 4 and 5 for preset fa and hl if they are of different |
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102 | 1 | Amber Herold | camera binning. |
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110 | h2. Presets setup and grid atlas |
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114 | 2 | Amber Herold | # Insert Grid. |
115 | # Make sure you're near eucentric height (z = 0 is close enough for Leginon, unless |
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116 | 1 | Amber Herold | your grid is seriously bent). |
117 | 2 | Amber Herold | # You should know from experience whether the imported "gr" preset aligns reasonable |
118 | 1 | Amber Herold | well to higher mags so that you can reach your intended grid square, however inaccurate, |
119 | without alignment. If this is true, you can follow 4 and 5 immediately. If not, you will |
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120 | need to manually move to a test sacrificial square at Tecnai and acquire the grid atlas |
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121 | after presets alignment. |
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122 | 2 | Amber Herold | # Leginon/Grid Targeting> Calculate and then publish a grid atlas using the "Grid |
123 | 1 | Amber Herold | Targeting" node. Do NOT pick targets in "Square" node during atlas assembly. |
124 | 2 | Amber Herold | # Leginon/Square Targeting> Pick an broken square on the grid atlas and press the |
125 | 1 | Amber Herold | play icon, then save position on Tecnai/TUI/Stage> or in Leginon/Navigation/Stage |
126 | Locations>. Repeat for a sacrificial square. The broken square is used to manually |
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127 | align the ZLP; the sacrificial square is used to align presets, objective lens, and to |
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128 | record test images. |
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136 | h2. Presets alignment |
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139 | 2 | Amber Herold | Note: steps 1 and 2 are only necessary for really bad grids (you'll know). |
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142 | 2 | Amber Herold | # Tecnai/TUI> stage: go to the sacrificial square. Turn on wobbler |
143 | # Tecnai/DM> do auto eucentric height and auto focus. |
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144 | # Leginon/Presets Manager> hl preset to scope. |
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145 | # Tecnai/TUI> move the stage to a feature recongnizable across the presets. |
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146 | # Leginon/Presets Manager> tomo preset to scope. |
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147 | # Tecnai/TUI> recenter the feature accurately. |
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148 | 3 | Amber Herold | # Leginon/Presets Manager> Follow [[MSI_set-up_in_more_details#Medium-mag-preset-image-and-beam-shift-refinement|image shift |
149 | 2 | Amber Herold | alignment procedure]] in general MSI set-up using tomo preset as the |
150 | 1 | Amber Herold | reference. |
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158 | h2. Objective aperture centering |
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162 | * Leginon/Presets Manager> tomography presets to scope. |
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165 | * Tecnai/TUI/motorized apertures> Put in objective aperture. |
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168 | * Tecnai> Go into diffraction mode. |
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171 | * Tecnai> Center image using diffraction shift-X&Y. |
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174 | * Tecnai/TUI/motorized apertures> Click center in motorized apertures menu. |
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177 | * Tecnai/TUI/motorized apertures> Center objective aperture, then remove objective |
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178 | aperture. |
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186 | h2. Target acquisition |
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190 | * Leginon/Grid Targeting> Remove objective aperture, Rename and calculate a new |
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191 | grid atlas, then publish to make a new atlas, and then Reinsert objective |
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192 | aperture. |
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195 | ***Note: You only need to take this (second) atlas if the presets - especially |
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196 | square and hole - are poorly aligned, which you'll find out from the previous |
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197 | alignments. If the presets were already aligned, then you can use the original grid |
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198 | atlas. *** |
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201 | * Leginon/Square Targeting> Click on the purple crosshair icon (reference point) |
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202 | and click on a broken square, which will be used for ZLP alignment and dosage. |
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205 | * Leginon/Square Targeting> Pick squares that look good, then press Play button |
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206 | !http://emg.nysbc.org/software/leginon/images/icons/play.png!. |
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209 | * Leginon/Hole Targeting> While square targets are getting collected, you can |
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210 | start picking targets in the hole targeting menu. Pick a bunch of holes (but not |
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211 | adjacent ones), then pick a focus point for rough Z- focus. To advance to the next |
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212 | square, press the play button but NOT Queue- play!http://emg.nysbc.org/software/leginon/images/icons/send_queue_out.png!. |
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215 | * Leginon/Hole Targeting> After the hole targets are all selected, insert the |
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216 | objective aperture and press Queue-play. |
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219 | * Leginon/Tomography Targeting> DON'T PRESS Queue-play until ready for final |
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220 | tomography acquisition. |
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223 | * Leginon/Tomography Targeting>Remember to pick a FOCUS spot for each hole and |
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224 | tomography target! |
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227 | Tip: you can mouse over the target and get an estimate of the average intensity in |
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228 | the popup menu, which lists X,Y,Cnts. This feature lets you rapidly screen for (1) |
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229 | squares with the thinnest ice and (2) the thinnest targets. |
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232 | * Leginon/Tomography Targeting> To preview target, use preview crosshair to pick |
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233 | the target: |
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234 | * SET defocus in preview preset to -1.2 e-06 m and adjust the exposure time (in |
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235 | milliseconds) such that the dose is ~ 0.2 - 0.5 e-/Angstrum^2 - VERY |
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236 | IMPORTANT! |
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239 | * If you pick a preview target and press Play button, it will be processed |
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240 | immediately. Therefore, don't do it until all hl images are taken. |
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243 | * After you have inspected the preview image, you can come back to Tomography |
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244 | Targeting node to pick final targets on the same image. Alternatively, you can |
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245 | pick the targets first with the preview target and then remove the unwanted ones |
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246 | after previewing. |
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252 | * Leginon/Tomography> check data collection parameters: dose, tilt range, tilt |
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253 | interval. See <link linkend="Tomo_Intensity">notes on image intensity recorded through |
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254 | tomography</link>. (If you want -60 to 60 tilts, you should ender -61 to 61 since the |
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255 | limiting algorithm is "smaller than" not "smaller than or equal to". |
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258 | * Leginon/Presets> setup data collection parameters for focus & tomography: |
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259 | mag, defocus, INT (INT for focus should be greater than for tomo). |
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267 | h2. Final checklist: Getting ready to collect |
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271 | * Leginon/Presets manager> tomo preset to scope. |
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274 | * Tecnai/TUI/Stage> Go to reference point. |
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277 | * Leginon/Tomography node> re-check dose and re-center beam. |
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280 | * Leginon/Exposure targeting> press queue-play. Atlas does NOT need to be |
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281 | reloaded. |
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291 | ______ |
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293 | [[Running the application|< Running the application]] | [[Leginon "MSI-Tomography" Application|Leginon "MSI-Tomography" Application ^]] |
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294 | |||
295 | ______ |