MSI set up in more details » History » Version 1
Amber Herold, 04/22/2010 05:03 PM
1 | 1 | Amber Herold | h1. MSI set-up in more details |
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2 | |||
3 | |||
4 | |||
5 | h2. Starting Up |
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6 | |||
7 | |||
8 | |||
9 | Set up is most stable with a good microscope alignment. |
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10 | |||
11 | |||
12 | |||
13 | |||
14 | * Initial alignment at the microscope See Chapter on <link |
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15 | linkend="microscope_setup">Microscope setup</link> for details. |
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16 | |||
17 | |||
18 | |||
19 | |||
20 | * scope> Insert specimen holder |
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21 | |||
22 | |||
23 | * scope> Put stage to eucenter and reset defocus in both HM mode AND LM |
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24 | mode |
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25 | |||
26 | |||
27 | * scope> Check gun tilt and shift |
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28 | |||
29 | |||
30 | * scope> Check rotation center and beam tilt pivot points in the HM |
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31 | mode |
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32 | |||
33 | |||
34 | * scope> Save the goniomenter position for the square with intact |
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35 | supporting film where the alignment was done and do the same for a square where |
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36 | the supporting film is broken (where bright/dark images can be acquired). |
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37 | |||
38 | |||
39 | |||
40 | |||
41 | |||
42 | |||
43 | * Start python-based leginon main program amd leginon |
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44 | client. See Chapter on <link linkend="microscope_setup">Start Leginon</link>. |
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45 | |||
46 | |||
47 | |||
48 | |||
49 | * scope> Set up <link linkend="CCDnote">CCD camera</link> |
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50 | |||
51 | |||
52 | * <link linkend="test_start_client">scope> Start Leginon Client</link> |
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53 | |||
54 | |||
55 | * scope> If the error message |
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56 | |||
57 | <blockquote> |
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58 | |||
59 | _"Exception EAccessViolation in module adaExp.exe at |
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60 | ...._ |
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61 | |||
62 | |||
63 | _Access violation at address xxxx in module 'adaExp.exe'. Read of |
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64 | address xxxx"_ |
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65 | |||
66 | </blockquote> |
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67 | appears, DO NOT touch it, or Leginon Client will need restarting |
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68 | |||
69 | |||
70 | * <link linkend="test_start_main">main> start-leginon.py</link> |
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71 | |||
72 | |||
73 | * Leginon Setup Wizard> Create new or Select old session, connect to the |
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74 | instrument host, and start. |
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75 | |||
76 | |||
77 | |||
78 | |||
79 | |||
80 | |||
81 | * Start your preferred "MSI" application. |
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82 | |||
83 | |||
84 | |||
85 | |||
86 | * Leginon/Application/Run> Select the "MSI" application you would like to |
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87 | use (Edge,T,Raster, or Tomography), the scope, and the main launcher. Click |
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88 | Run. |
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89 | |||
90 | |||
91 | * Leginon/Node Selector> Select "Presets Manager" node. |
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92 | |||
93 | |||
94 | * Leginon/Presets Manager/Preset Selector> Import |
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95 | presets from a previous working session <inlinegraphic |
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96 | fileref="http://emg.nysbc.org/software/leginon/images/icons/import.png" |
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97 | format="PNG"/> |
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98 | |||
99 | |||
100 | * Select the TEM and camera to "Find" sessions closest to the condition of |
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101 | your data collection. |
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102 | |||
103 | |||
104 | * Use shift-key to select a series of presets if needed. Hold-down to |
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105 | ctrl-key allows individual selection/deselection. |
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106 | |||
107 | |||
108 | * Click "Import" button to import, the presets should appear in the |
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109 | Presets Manager/Preset Selector window. |
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110 | |||
111 | |||
112 | * Click "OK" to exit "Import Presets" window. |
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113 | |||
114 | |||
115 | |||
116 | * Creating new preset and adjusting the preset are covered in Calibration |
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117 | Chapter under <link linkend="presets_create">Creating presets the first |
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118 | time</link>. |
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119 | |||
120 | |||
121 | * The cycle order should match the sequence of data acquisition to minimize |
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122 | hysterisis. If the presets are imported from previous session, the cycle order |
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123 | is imported automatically. As an example, the cycle for naming scheme used here |
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124 | at NRAMM is gr -> sq -> hl -> fa -> fc -> en -> ef. |
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125 | |||
126 | |||
127 | * Leginon/Presets Manager> Select a preset in |
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128 | the list box and then click "To Scope" <inlinegraphic |
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129 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
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130 | format="PNG"/> to apply the settings to the scope and synchronize Leginon and |
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131 | the scope settings. |
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132 | |||
133 | |||
134 | |||
135 | |||
136 | |||
137 | |||
138 | * Save the alignment and empty position in Leginon |
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139 | |||
140 | |||
141 | |||
142 | |||
143 | * scope> Recall the stored intact or empty position or go to such |
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144 | location. |
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145 | |||
146 | |||
147 | * Leginon/Navigation/Toolbar/Stage Locations> |
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148 | <inlinegraphic |
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149 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
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150 | format="PNG"/> Enter a New name to store Stage Location such as, for example, |
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151 | "empty" and "align", respectively. Save the xy coordinate only. To recall the |
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152 | position, select the named location under the same tool and click on "send to |
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153 | scope". |
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154 | |||
155 | |||
156 | |||
157 | |||
158 | |||
159 | |||
160 | |||
161 | |||
162 | |||
163 | |||
164 | |||
165 | |||
166 | |||
167 | h2. Grid preset image shift refinement |
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168 | |||
169 | |||
170 | |||
171 | |||
172 | |||
173 | * Leginon/Presets Manager> Send a preset at an intermediate mag at HM mode as |
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174 | the reference (e.g. hl) "To Scope"<inlinegraphic |
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175 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
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176 | format="PNG"/> |
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177 | |||
178 | |||
179 | (Note: sq preset in LM mode can be used if the image alignment between LM and HM |
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180 | is known to be good). |
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181 | |||
182 | |||
183 | * scope> Move the grid to a position on the "align" square, either on a |
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184 | recognizable object or burn a good hole through ice at the center of the CCD as |
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185 | preparation for lower mag preset image shift correction. |
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186 | |||
187 | |||
188 | * Leginon/Presets Manager> Click on the tool "Align presets to each other" |
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189 | <inlinegraphic |
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190 | fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png" |
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191 | format="PNG"/> . |
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192 | |||
193 | |||
194 | * Check, adjust, and save the preset(s) to be aligned. |
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195 | |||
196 | |||
197 | The left panel displays and navigate by "Stage Position" of the reference preset |
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198 | while the right panel displays and navigate by "Image Shift" of the preset to be |
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199 | aligned |
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200 | * Leginon/Presets Manager/Align Presets> Choose the reference (hl) preset |
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201 | on the left panel. |
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202 | |||
203 | |||
204 | * Leginon/Presets Manager/Align Presets> Choose the aligning (gr) preset |
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205 | on the right panel. |
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206 | |||
207 | |||
208 | * Leginon/Presets Manager/Align Presets> Select "Similar look across mags" |
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209 | mode at the top left corner of the window. This will use a smaller part of the |
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210 | CCD to acquire the image for the lower mag preset so that it appears to be |
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211 | magnified in the 512x512 image panel. |
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212 | |||
213 | |||
214 | * Leginon/Presets Manager/Align Presets> "Start" image acquisition. |
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215 | |||
216 | |||
217 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
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218 | not be found in both panels, go back to step one to choose a lower mag preset or |
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219 | a better object. |
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220 | |||
221 | |||
222 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
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223 | be found in both panels but not at the center on the reference left panel, |
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224 | navigate* it to the center using its "Click Tool". Wait for the move to complete |
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225 | and both images refreshed. |
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226 | |||
227 | |||
228 | * Leginon/Presets Manager/Align Presets> Navigate* the object on the right |
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229 | panel to the center using its "Click Tool".The new image shift is automatically |
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230 | saved at each move. |
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231 | |||
232 | |||
233 | * Repeat 7 if necessary. |
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234 | |||
235 | |||
236 | |||
237 | |||
238 | |||
239 | |||
240 | |||
241 | |||
242 | |||
243 | * "Navigate" means selecting the navigate tool<inlinegraphic |
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244 | fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png" format="PNG"/> on |
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245 | the top right of image display and then double clicking the location of the new center that |
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246 | is translated by the given TEM Parameter. |
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247 | |||
248 | |||
249 | |||
250 | |||
251 | |||
252 | h2. Grid preset stage postion refinement |
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253 | |||
254 | |||
255 | |||
256 | Stage position model Scale and Rotation is stable as long as the LM eucentric height |
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257 | defocus is set to zero before each session, although variation from day to day is normal. |
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258 | Check the stage movement calibration of gr preset by navigating it in Navigation node. |
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259 | Within 10% error is satisfactory since reaching a square does not require high accuracy. If |
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260 | not, perform stage model position _<link linkend="gonm_m_only">scale and rotation |
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261 | calibration</link> at gr preset_ using "Calibration" application as |
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262 | following: |
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263 | |||
264 | |||
265 | _<link linkend="gonmodel_how">Why goniometer model is |
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266 | better</link>._ |
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267 | |||
268 | |||
269 | |||
270 | |||
271 | * scope> Remove the objective apperture. |
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272 | |||
273 | |||
274 | * Leginon> Run "Calibrations" Application |
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275 | |||
276 | |||
277 | * Leginon/Presets Manager> Send "gr" preset to scope. |
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278 | |||
279 | |||
280 | * Leginon/GonioModeling/Settings/Measurement> Set axis=x, |
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281 | Points=5,Tolerance=25%, and Interval=1e-05 m. Default model label is the session |
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282 | name. |
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283 | |||
284 | |||
285 | * Leginon/GonioModeling/Settings/Modeling> Set "Scale and Rotation Adjustment |
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286 | Only"=Yes. Other settings will be set automatically once measurements are |
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287 | finished. |
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288 | |||
289 | |||
290 | * Leginon/GonioModeling> Click on <inlinegraphic |
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291 | fileref="http://emg.nysbc.org/software/leginon/images/icons/cam_ruler.png" |
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292 | format="PNG"/> will start "Measure" the shifts of between points that are moved on x |
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293 | axis of the goniometer. |
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294 | |||
295 | |||
296 | * Leginon/GonioModeling> Click on <inlinegraphic |
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297 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG" |
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298 | /> will "Calibrate" the scale and rotation of the model on x axis of the |
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299 | goniometer. |
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300 | |||
301 | |||
302 | * Leginon/GonioModeling> repeat 4-7 with the axis set to y. |
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303 | |||
304 | |||
305 | |||
306 | |||
307 | |||
308 | |||
309 | |||
310 | |||
311 | |||
312 | h2. Acquire mosaic atlas of the grid |
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313 | |||
314 | |||
315 | |||
316 | |||
317 | |||
318 | * scope> Remove the objective apperture. |
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319 | |||
320 | |||
321 | * Leginon/Grid Targeting> Click "Settings" to check the preset used, the size |
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322 | and name of the atlas. |
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323 | |||
324 | |||
325 | * Leginon/Grid Targeting> Click "Calculate Atlas" <inlinegraphic |
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326 | fileref="http://emg.nysbc.org/software/leginon/images/icons/calculate.png" |
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327 | format="PNG"/> |
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328 | |||
329 | |||
330 | * Leginon/Grid Targeting> Click "Submit Targets" <inlinegraphic |
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331 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG" |
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332 | />to begin acquiring the atlas. |
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333 | |||
334 | |||
335 | * Leginon/Square Targeting> Follow the atlas progress in the display. (***There |
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336 | is no sign at the end currently other than that nothing happens for a long time and |
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337 | that the atlas dimension is the same in other direction when it is completed.) |
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338 | |||
339 | |||
340 | * scope> Insert the objective aperture. |
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341 | |||
342 | |||
343 | * Leginon/Presets Manager/Send an HM preset "To Scope" several times to cycle all |
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344 | magnifications and warm up the objective lens to minimize the image shift hysteresis |
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345 | in switching between LM and HM modes. If the scope has been in LM mode for a long |
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346 | time, wait for at least 30 min before final image shift refinement of needed LM preset |
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347 | during acquisition (i.e. sq) |
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348 | |||
349 | |||
350 | |||
351 | |||
352 | |||
353 | |||
354 | |||
355 | |||
356 | |||
357 | h2. Determine the proper exposure condition for exposure |
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358 | presets |
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359 | |||
360 | |||
361 | |||
362 | Exposure presets are the base presets for data collection. To keep it to low dose |
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363 | condition such as 10 electron/Angstrum, measurement of dose is performed and the exposure |
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364 | time is adjusted. If scope or camera parameters are changed during the session, the dose |
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365 | value is no longer valid and should be remeasured. |
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366 | |||
367 | |||
368 | |||
369 | |||
370 | * Leginon/Navigation/Toolbar/Stage Locations<inlinegraphic |
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371 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
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372 | format="PNG"/>> Send the "empty" position "To scope". |
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373 | |||
374 | |||
375 | * Leginon/Presets Manager/Preset Selector> Select a high mag exposure (i.e. "en" |
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376 | or "ef"), confirm that the parameters are what you desire for the experiment, and then |
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377 | send "To Scope"<inlinegraphic |
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378 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
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379 | format="PNG"/>. |
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380 | |||
381 | |||
382 | * scope> Expand the beam to at least cover the CCD (> medium circle mark on |
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383 | the viewing screen) and to the size you desire. |
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384 | |||
385 | |||
386 | * Leginon/Presets Manager/Preset Selector> If you have changed the size or |
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387 | location of the beam or other preset scope parameters at the scope, you must retrieve |
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388 | the preset parameter "From scope"<inlinegraphic |
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389 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png" |
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390 | format="PNG"/> first before checking and saving the dose value. |
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391 | |||
392 | |||
393 | * Leginon/Presets Manager/Preset Selector> "Acquire dose image"<inlinegraphic |
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394 | fileref="http://emg.nysbc.org/software/leginon/images/icons/acquire.png" |
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395 | format="PNG"/> for the selected preset. |
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396 | |||
397 | |||
398 | * Leginon/Presets Manager/Dose Image> Check the "Dose" value. |
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399 | |||
400 | |||
401 | * Leginon/Presets Manager/Dose Image> If desired dose is known, fill in the |
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402 | value and then click "Match". This will change exposure time to give a close match of |
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403 | the value entered. |
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404 | |||
405 | |||
406 | * Leginon/Presets Manager/Dose Image> If not satisfied with the value, click |
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407 | "No", change the exposure time by editing the preset, and try again (No need to send |
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408 | the preset "To scope" again for the repeat) |
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409 | |||
410 | |||
411 | * If the scope can not give such illumination to satisfy dose, exposure time and |
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412 | area requirement, alignment is required or change of gun lens or spot size is |
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413 | needed. |
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414 | |||
415 | |||
416 | * Leginon/Presets Manager> Copy the preset parameter to the other exposure |
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417 | preset. |
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418 | |||
419 | |||
420 | * Preset Selector> send the preset that has the dose measurement "To |
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421 | Scope"<inlinegraphic |
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422 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
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423 | format="PNG"/> |
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424 | |||
425 | |||
426 | * Preset Selector> select the preset to recieve the copy |
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427 | |||
428 | |||
429 | * Preset Selector> retrieve the parameters "From Scope"<inlinegraphic |
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430 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png" |
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431 | format="PNG"/> for the selected preset. |
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432 | |||
433 | |||
434 | * Preset Selector/Edit><inlinegraphic |
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435 | fileref="http://emg.nysbc.org/software/leginon/images/icons/edit.png" |
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436 | format="PNG"/> Modify defocus for the preset after the retrieval. |
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437 | |||
438 | |||
439 | |||
440 | |||
441 | |||
442 | |||
443 | |||
444 | |||
445 | |||
446 | |||
447 | |||
448 | h2. Update bright/dark correction images for exposure |
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449 | presets |
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450 | |||
451 | |||
452 | |||
453 | Exposure presets are the base presets for data collection. To make sure the best |
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454 | correction is applied to them, the correction images should be acquired prior to the |
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455 | experiment at session. Since no correlation is normally done on these images, only one <link |
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456 | linkend="Cal_cor_channel">channel</link> is needed. Normalization image might need to be |
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457 | <link linkend="Cal_cor_region">modified</link> if a large chunk of region is blocked by a |
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458 | fallen debris. |
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459 | |||
460 | |||
461 | |||
462 | |||
463 | * Leginon/Presets Manager> Select a dose measured high mag exposure (i.e. "en" |
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464 | or "ef"), and then send "To Scope". |
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465 | |||
466 | |||
467 | * Leginon/Correction/Settings> |
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468 | |||
469 | |||
470 | * Select TEM and camera to match that will be used. |
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471 | |||
472 | |||
473 | * Camera Configuration: The exposure preset camera configuration |
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474 | |||
475 | |||
476 | |||
477 | |||
478 | * Leginon/Correction/Settings> Click OK to exit settings. |
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479 | |||
480 | |||
481 | * Leginon/Correction/Toolbar> Select "Raw image " from the pull down list of |
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482 | acquisition modes and then click on "Acquire" button next to the selector to view an |
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483 | image that is not corrected. |
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484 | |||
485 | |||
486 | * Leginon/Correction/Toolbar> Select "Dark reference" in the acquisition mode |
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487 | selector and then click "Acquire" to acquire the Dark reference image for this |
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488 | particular camera configuration. |
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489 | |||
490 | |||
491 | * Leginon/Correction/Toolbar> Select "Bright reference" and repeat the |
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492 | acquisition to obtain the Bright reference. |
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493 | |||
494 | |||
495 | * Leginon/Correction/Toolbar> Select "Corrected image" and then "Acquire" to |
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496 | view the corrected image. A corrected image should be free of artifacts and have |
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497 | smaller standard deviation than the raw image, in general. |
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498 | |||
499 | |||
500 | * Leginon/Correction/Toolbar> If there are regions consistently over normalizes |
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501 | the acquired image, either select the region and use the "+" tool to "Add Region to |
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502 | Bad Pixel List", or click on<inlinegraphic |
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503 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
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504 | format="PNG"/>to "Add Extreme Points to Bad Pixel List" one of more times to find |
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505 | the outliers. Right click on a shown bad pixel removes it from the list. |
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506 | |||
507 | |||
508 | |||
509 | |||
510 | |||
511 | |||
512 | |||
513 | |||
514 | |||
515 | h2. Medium mag preset image and beam shift |
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516 | refinement |
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517 | |||
518 | |||
519 | |||
520 | Preset image shift alignment can be done follow the same procedure as the section "<link |
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521 | linkend="image_shift_align">Grid preset image shift refinement</link>" but is more |
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522 | efficient using the automated mode as follows. |
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523 | |||
524 | |||
525 | |||
526 | |||
527 | * Leginon/Presets Manager> Send one of the exposure preset as the reference |
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528 | (e.g. en) "To Scope"<inlinegraphic |
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529 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
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530 | format="PNG"/> |
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531 | |||
532 | |||
533 | * scope> Move the grid to a position on the "align" square, either on a |
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534 | recognizable object or burn a good hole through ice at the center of the CCD as |
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535 | preparation for lower mag preset image shift correction. |
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536 | |||
537 | |||
538 | * Leginon/Presets Manager> Click on the tool "Align presets to each other" |
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539 | <inlinegraphic |
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540 | fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png" |
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541 | format="PNG"/> . The tool automatically select the reference preset that was |
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542 | selected in the Presets Manager mainpanel and choose a preset at closest magnification |
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543 | as the starting aligning preset. |
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544 | |||
545 | |||
546 | * Leginon/Presets Manager/Align Presets> Click "Start" to acquire the first |
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547 | image pair. |
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548 | |||
549 | |||
550 | * Check, adjust, and save the preset(s) to be aligned. |
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551 | |||
552 | |||
553 | The left panel displays and navigate by "Stage Position" of the reference preset |
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554 | while the right panel displays and navigate by "Image Shift" of the preset to be |
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555 | aligned |
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556 | * Leginon/Presets Manager/Align Presets> Changing Selection of "Similar |
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557 | look across mags" or "Full CCD" mode at the top left corner of the window will |
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558 | cause reacquisition of the image pair in the automated mode. |
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559 | |||
560 | |||
561 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
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562 | be found in both panels but not at the center on the reference left panel, |
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563 | navigate* it to the center using its "Click Tool". Wait for the move to complete |
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564 | and both images refreshed. |
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565 | |||
566 | |||
567 | * Leginon/Presets Manager/Align Presets> Navigate* the object on the right |
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568 | panel to the center using its "Click Tool". The new image shift is automatically |
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569 | saved after each move. |
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570 | |||
571 | |||
572 | * Repeat 3 if necessary. |
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573 | |||
574 | |||
575 | * Leginon/Presets Manager/Align Presets> Click "Continue" to save the |
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576 | alignment to all presets of the same magnification and continue to the next |
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577 | preset pair. |
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578 | |||
579 | |||
580 | |||
581 | |||
582 | |||
583 | * Repeat the process if so desired. |
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584 | |||
585 | |||
586 | |||
587 | |||
588 | |||
589 | |||
590 | |||
591 | |||
592 | |||
593 | h2. Go to MSI Operation section |
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594 | |||
595 | |||
596 | |||
597 | The setup that requires you to be next to the microscope is completed. Go to the section |
||
598 | on <link linkend="leg1operation">MSI Operation</link>. |
||
599 | |||
600 | |||
601 | |||
602 | |||
603 | |||
604 | h2. Hole Targeting Set-up |
||
605 | |||
606 | |||
607 | |||
608 | We can use any image that is already loaded into Hole Targeting to adjust these |
||
609 | parameters. The values in this node vary so much from grid to grid that the best way to |
||
610 | find these parameters is by going through a step-by-step trial-and-error process. To see the |
||
611 | final acquisition and focus targets, enable only the Original, acquisition, and focus |
||
612 | display selections. Refer to Introduction Chapter on <link linkend="image_viewer_control" |
||
613 | >how the various tools behave in the control panel</link>. |
||
614 | |||
615 | |||
616 | The loaded image normally comes from the image acquired through "Square" node when you |
||
617 | submit targets on the grid atlas in "Square Targeting" node. You can also do a practice run |
||
618 | using the default image comes with Leginon. Leave the filename entry blank when you click |
||
619 | "Load" in the hole finding settings window for the step "Original". The default values in |
||
620 | the node gives an example of reasonable target finding with such default image. |
||
621 | |||
622 | |||
623 | |||
624 | |||
625 | Setting up Hole Targeting Depends on which MSI flavor you are running: |
||
626 | |||
627 | |||
628 | |||
629 | |||
630 | * <link linkend="Eholetargeting_setup">MSI-Edge</link> uses template matching of the |
||
631 | edge image |
||
632 | |||
633 | |||
634 | * <link linkend="MSIT_tmplt_cor">MSI-T</link> uses template matching of the original |
||
635 | image |
||
636 | |||
637 | |||
638 | * <link linkend="Rholetarget_setup">MSI-Raster</link> creates raster points from a given |
||
639 | origin |
||
640 | |||
641 | |||
642 | * <link linkend="TomoTargeting_setup">MSI-Tomography</link> does not use automatic |
||
643 | target finding. Therefore, it does not requires set up. |
||
644 | |||
645 | |||
646 | |||
647 | |||
648 | |||
649 | |||
650 | |||
651 | |||
652 | |||
653 | h2. Exposure Targeting Set-up |
||
654 | |||
655 | |||
656 | |||
657 | Like hole targeting, we can use any image that is already loaded into Exposure Targeting |
||
658 | to adjust these parameters. |
||
659 | |||
660 | Setting up Exposure Targeting Depends on which MSI flavor you are running. |
||
661 | |||
662 | |||
663 | * <link linkend="Eexptargeting_setup">MSI-Edge</link> uses template matching of the edge |
||
664 | image |
||
665 | |||
666 | |||
667 | * <link linkend="MSIT_tmplt_cor">MSI-T</link> uses template matching of the original |
||
668 | image |
||
669 | |||
670 | |||
671 | * <link linkend="Rexptarget_setup">MSI-Raster</link> creates raster points from a given |
||
672 | origin |
||
673 | |||
674 | |||
675 | * <link linkend="TomoTargeting_setup">MSI-Tomography</link> does not use automatic target |
||
676 | finding. Therefore, it does not require set up. |
||
677 | |||
678 | |||
679 | |||
680 | |||
681 | |||
682 | |||
683 | |||
684 | h2. Autofocus Test |
||
685 | |||
686 | |||
687 | |||
688 | There are two focusers in MSI. "Z Focus" node usually moves stage in Z direction to |
||
689 | Eucentric height at lower mags. "Focus" node usually just correct the defocus in the way |
||
690 | equivalent to turning the focus adjustment knob on the scope at a higher mag. The defocus |
||
691 | measurments in both nodes are based on the same principle of beam-tilt induced image shift. |
||
692 | A correlation (normally phase) is used to obtain the image shift. Therefore, it is important |
||
693 | to test and preform autofocus measurement at area with recognizable feature that does not |
||
694 | move significantly during the defocus measurement. |
||
695 | |||
696 | |||
697 | The test use "Manual_after" focus step in the focus sequence for checking. Two |
||
698 | procedures can be used to reach a area for good correlation measurements: |
||
699 | |||
700 | |||
701 | |||
702 | |||
703 | * Using Simulate Target tool |
||
704 | |||
705 | |||
706 | |||
707 | |||
708 | * Leginon/Presets Manager> Send the autofocus preset "To Scope" |
||
709 | |||
710 | |||
711 | * scope> Move to an area that has recognizable feature on the carbon |
||
712 | support at the center. |
||
713 | |||
714 | |||
715 | * Leginon/(Z) Focus/Focus Sequence<inlinegraphic |
||
716 | fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png" |
||
717 | format="PNG"/>> Enable desired autofocus and "Manual_after" focus |
||
718 | steps. |
||
719 | |||
720 | |||
721 | * Leginon/(Z) Focus/Toolbar> Click Simulate Target<inlinegraphic |
||
722 | fileref="http://emg.nysbc.org/software/leginon/images/icons/simulatetarget.png" |
||
723 | format="PNG"/> and follow the autofocus |
||
724 | |||
725 | |||
726 | |||
727 | |||
728 | |||
729 | |||
730 | * Submiting focus target from Hole Targeting |
||
731 | |||
732 | |||
733 | *To use this procedure, there has to be a grid mosaic, and the Hole Targeting |
||
734 | should allow user verification of targets |
||
735 | |||
736 | |||
737 | * Leginon/(Z) Focus/Focus Sequence<inlinegraphic |
||
738 | fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png" |
||
739 | format="PNG"/>> Enable desired autofocus and "Manual_after" focus |
||
740 | steps. |
||
741 | |||
742 | |||
743 | * Leginon/Square Targeting> Select a test square and submit it<inlinegraphic |
||
744 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" |
||
745 | format="PNG"/> |
||
746 | |||
747 | |||
748 | * Leginon/Hole Targeting> Submit a focus target to test autofocus in Z Focus |
||
749 | node and an acquisition target for the next stage. Both targets should be in an |
||
750 | area that has recognizable feature with carbon support. |
||
751 | |||
752 | |||
753 | * Leginon/Z focus> follow the autofocus test in this node. |
||
754 | |||
755 | |||
756 | * Leginon/Exposure Targeting> Submit a focus target at a location that has |
||
757 | recognizable feature with carbon support to test autofocus in Focus node. |
||
758 | |||
759 | |||
760 | * Leginon/Focus> follow autofocus test in this node. |
||
761 | |||
762 | |||
763 | |||
764 | |||
765 | |||
766 | |||
767 | |||
768 | |||
769 | Checking the performance of autofocus by the following |
||
770 | criteria: |
||
771 | |||
772 | |||
773 | |||
774 | |||
775 | * During autofocusing |
||
776 | |||
777 | |||
778 | |||
779 | |||
780 | * Display the image and correlection peak: The observed movement of images |
||
781 | upon beam tilt should be consistent with the correlation peak and the edge of |
||
782 | the beam should not be visible in any of the beam-tilt images. |
||
783 | |||
784 | |||
785 | * Display the correlation image: The correlation peak at its correct position |
||
786 | should be significantly higher than background. |
||
787 | |||
788 | |||
789 | |||
790 | |||
791 | |||
792 | |||
793 | * During manual focusing |
||
794 | |||
795 | Manual Focusing can be activated either by enabling "Manual_before/Manual_after" |
||
796 | steps or directly when leginon is idle using (Z) Focus/Toolbar/MF. To activate manual |
||
797 | focus directly, it is necessary to send to scope the preset of the choice (normally |
||
798 | fc) first, though. |
||
799 | |||
800 | |||
801 | * MF> The FFT should show visible and isotropic Thon ring according to the |
||
802 | fc preset defocus. |
||
803 | |||
804 | |||
805 | * MF/Toolbar> Make sure the move type is "Defocus" and the set value in the |
||
806 | box is 0 m then click "Set instrument" botton<inlinegraphic |
||
807 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
808 | format="PNG"/>. This will set the defocus to the current 0. |
||
809 | |||
810 | |||
811 | * MF> The perfect autofocus result is that the FFT indicates that the scope |
||
812 | is at Gaussian focus. |
||
813 | |||
814 | |||
815 | * For relative movement, press "+" or "-". The step size is determined in |
||
816 | MF/Toolbar/Settings> |
||
817 | |||
818 | |||
819 | * To reset defocus to zero at the new focus, press the so-named |
||
820 | button<inlinegraphic |
||
821 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentsetnew.png" |
||
822 | format="PNG"/>. |
||
823 | |||
824 | |||
825 | * Clicking the Stop button<inlinegraphic |
||
826 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stop.png" |
||
827 | format="PNG"/> exits the maunal check. |