MSI set up in more details » History » Version 4
Alex Kadokura, 06/04/2010 03:53 PM
1 | 1 | Amber Herold | h1. MSI set-up in more details |
---|---|---|---|
2 | |||
3 | |||
4 | |||
5 | h2. Starting Up |
||
6 | |||
7 | |||
8 | |||
9 | Set up is most stable with a good microscope alignment. |
||
10 | |||
11 | |||
12 | |||
13 | |||
14 | 3 | Alex Kadokura | * Initial alignment at the microscope See Chapter on [[Microscope Set-up|Microscope setup]] for details. |
15 | 1 | Amber Herold | |
16 | |||
17 | |||
18 | |||
19 | * scope> Insert specimen holder |
||
20 | |||
21 | |||
22 | * scope> Put stage to eucenter and reset defocus in both HM mode AND LM |
||
23 | mode |
||
24 | |||
25 | |||
26 | * scope> Check gun tilt and shift |
||
27 | |||
28 | |||
29 | * scope> Check rotation center and beam tilt pivot points in the HM |
||
30 | mode |
||
31 | |||
32 | |||
33 | * scope> Save the goniomenter position for the square with intact |
||
34 | supporting film where the alignment was done and do the same for a square where |
||
35 | the supporting film is broken (where bright/dark images can be acquired). |
||
36 | |||
37 | |||
38 | |||
39 | |||
40 | |||
41 | |||
42 | * Start python-based leginon main program amd leginon |
||
43 | 3 | Alex Kadokura | client. See Chapter on [[Microscope Set-up|Start Leginon]]. |
44 | 1 | Amber Herold | |
45 | |||
46 | |||
47 | |||
48 | 3 | Alex Kadokura | * scope> Set up [[MSI set-up in more details#Starting-Up|CCD camera]] |
49 | 1 | Amber Herold | |
50 | 3 | Alex Kadokura | * [[Run Leginon Client on the Microscope Computer|scope> Start Leginon Client]] |
51 | 1 | Amber Herold | |
52 | |||
53 | * scope> If the error message |
||
54 | |||
55 | <blockquote> |
||
56 | |||
57 | _"Exception EAccessViolation in module adaExp.exe at |
||
58 | ...._ |
||
59 | |||
60 | |||
61 | _Access violation at address xxxx in module 'adaExp.exe'. Read of |
||
62 | address xxxx"_ |
||
63 | |||
64 | </blockquote> |
||
65 | appears, DO NOT touch it, or Leginon Client will need restarting |
||
66 | |||
67 | |||
68 | 3 | Alex Kadokura | * [[Start Leginon at your main working station remotely|main> start-leginon.py]] |
69 | 1 | Amber Herold | |
70 | |||
71 | * Leginon Setup Wizard> Create new or Select old session, connect to the |
||
72 | instrument host, and start. |
||
73 | |||
74 | |||
75 | |||
76 | |||
77 | |||
78 | |||
79 | * Start your preferred "MSI" application. |
||
80 | |||
81 | |||
82 | |||
83 | |||
84 | * Leginon/Application/Run> Select the "MSI" application you would like to |
||
85 | use (Edge,T,Raster, or Tomography), the scope, and the main launcher. Click |
||
86 | Run. |
||
87 | |||
88 | |||
89 | * Leginon/Node Selector> Select "Presets Manager" node. |
||
90 | |||
91 | |||
92 | * Leginon/Presets Manager/Preset Selector> Import |
||
93 | presets from a previous working session <inlinegraphic |
||
94 | fileref="http://emg.nysbc.org/software/leginon/images/icons/import.png" |
||
95 | format="PNG"/> |
||
96 | |||
97 | |||
98 | * Select the TEM and camera to "Find" sessions closest to the condition of |
||
99 | your data collection. |
||
100 | |||
101 | |||
102 | * Use shift-key to select a series of presets if needed. Hold-down to |
||
103 | ctrl-key allows individual selection/deselection. |
||
104 | |||
105 | |||
106 | * Click "Import" button to import, the presets should appear in the |
||
107 | Presets Manager/Preset Selector window. |
||
108 | |||
109 | |||
110 | * Click "OK" to exit "Import Presets" window. |
||
111 | |||
112 | |||
113 | |||
114 | * Creating new preset and adjusting the preset are covered in Calibration |
||
115 | 3 | Alex Kadokura | Chapter under [[Creating Presets for the first time|Creating presets the first |
116 | time]]. |
||
117 | 1 | Amber Herold | |
118 | |||
119 | * The cycle order should match the sequence of data acquisition to minimize |
||
120 | hysterisis. If the presets are imported from previous session, the cycle order |
||
121 | is imported automatically. As an example, the cycle for naming scheme used here |
||
122 | at NRAMM is gr -> sq -> hl -> fa -> fc -> en -> ef. |
||
123 | |||
124 | |||
125 | * Leginon/Presets Manager> Select a preset in |
||
126 | the list box and then click "To Scope" <inlinegraphic |
||
127 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
128 | format="PNG"/> to apply the settings to the scope and synchronize Leginon and |
||
129 | the scope settings. |
||
130 | |||
131 | |||
132 | |||
133 | |||
134 | |||
135 | |||
136 | * Save the alignment and empty position in Leginon |
||
137 | |||
138 | |||
139 | |||
140 | |||
141 | * scope> Recall the stored intact or empty position or go to such |
||
142 | location. |
||
143 | |||
144 | |||
145 | * Leginon/Navigation/Toolbar/Stage Locations> |
||
146 | <inlinegraphic |
||
147 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
||
148 | format="PNG"/> Enter a New name to store Stage Location such as, for example, |
||
149 | "empty" and "align", respectively. Save the xy coordinate only. To recall the |
||
150 | position, select the named location under the same tool and click on "send to |
||
151 | scope". |
||
152 | |||
153 | |||
154 | |||
155 | |||
156 | |||
157 | |||
158 | |||
159 | |||
160 | |||
161 | |||
162 | |||
163 | |||
164 | |||
165 | h2. Grid preset image shift refinement |
||
166 | |||
167 | |||
168 | |||
169 | |||
170 | |||
171 | * Leginon/Presets Manager> Send a preset at an intermediate mag at HM mode as |
||
172 | the reference (e.g. hl) "To Scope"<inlinegraphic |
||
173 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
174 | format="PNG"/> |
||
175 | |||
176 | |||
177 | (Note: sq preset in LM mode can be used if the image alignment between LM and HM |
||
178 | is known to be good). |
||
179 | |||
180 | |||
181 | * scope> Move the grid to a position on the "align" square, either on a |
||
182 | recognizable object or burn a good hole through ice at the center of the CCD as |
||
183 | preparation for lower mag preset image shift correction. |
||
184 | |||
185 | |||
186 | * Leginon/Presets Manager> Click on the tool "Align presets to each other" |
||
187 | <inlinegraphic |
||
188 | fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png" |
||
189 | format="PNG"/> . |
||
190 | |||
191 | |||
192 | * Check, adjust, and save the preset(s) to be aligned. |
||
193 | |||
194 | |||
195 | The left panel displays and navigate by "Stage Position" of the reference preset |
||
196 | while the right panel displays and navigate by "Image Shift" of the preset to be |
||
197 | aligned |
||
198 | * Leginon/Presets Manager/Align Presets> Choose the reference (hl) preset |
||
199 | on the left panel. |
||
200 | |||
201 | |||
202 | * Leginon/Presets Manager/Align Presets> Choose the aligning (gr) preset |
||
203 | on the right panel. |
||
204 | |||
205 | |||
206 | * Leginon/Presets Manager/Align Presets> Select "Similar look across mags" |
||
207 | mode at the top left corner of the window. This will use a smaller part of the |
||
208 | CCD to acquire the image for the lower mag preset so that it appears to be |
||
209 | magnified in the 512x512 image panel. |
||
210 | |||
211 | |||
212 | * Leginon/Presets Manager/Align Presets> "Start" image acquisition. |
||
213 | |||
214 | |||
215 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
||
216 | not be found in both panels, go back to step one to choose a lower mag preset or |
||
217 | a better object. |
||
218 | |||
219 | |||
220 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
||
221 | be found in both panels but not at the center on the reference left panel, |
||
222 | navigate* it to the center using its "Click Tool". Wait for the move to complete |
||
223 | and both images refreshed. |
||
224 | |||
225 | |||
226 | * Leginon/Presets Manager/Align Presets> Navigate* the object on the right |
||
227 | panel to the center using its "Click Tool".The new image shift is automatically |
||
228 | saved at each move. |
||
229 | |||
230 | |||
231 | * Repeat 7 if necessary. |
||
232 | |||
233 | |||
234 | |||
235 | |||
236 | |||
237 | |||
238 | |||
239 | |||
240 | |||
241 | * "Navigate" means selecting the navigate tool<inlinegraphic |
||
242 | fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png" format="PNG"/> on |
||
243 | the top right of image display and then double clicking the location of the new center that |
||
244 | is translated by the given TEM Parameter. |
||
245 | |||
246 | |||
247 | |||
248 | |||
249 | |||
250 | h2. Grid preset stage postion refinement |
||
251 | |||
252 | |||
253 | |||
254 | Stage position model Scale and Rotation is stable as long as the LM eucentric height |
||
255 | defocus is set to zero before each session, although variation from day to day is normal. |
||
256 | Check the stage movement calibration of gr preset by navigating it in Navigation node. |
||
257 | Within 10% error is satisfactory since reaching a square does not require high accuracy. If |
||
258 | 3 | Alex Kadokura | not, perform stage model position _[[Modeled Stage Position calibration#Magnification-and-image-rotation-adjustment-of-the-model|scale and rotation |
259 | calibration]]> at gr preset_ using "Calibration" application as |
||
260 | 1 | Amber Herold | following: |
261 | |||
262 | |||
263 | 3 | Alex Kadokura | _[[Modeled Stage Position calibration|Why goniometer model is |
264 | better]]._ |
||
265 | 1 | Amber Herold | |
266 | |||
267 | |||
268 | |||
269 | * scope> Remove the objective apperture. |
||
270 | |||
271 | |||
272 | * Leginon> Run "Calibrations" Application |
||
273 | |||
274 | |||
275 | * Leginon/Presets Manager> Send "gr" preset to scope. |
||
276 | |||
277 | |||
278 | * Leginon/GonioModeling/Settings/Measurement> Set axis=x, |
||
279 | Points=5,Tolerance=25%, and Interval=1e-05 m. Default model label is the session |
||
280 | name. |
||
281 | |||
282 | |||
283 | * Leginon/GonioModeling/Settings/Modeling> Set "Scale and Rotation Adjustment |
||
284 | Only"=Yes. Other settings will be set automatically once measurements are |
||
285 | finished. |
||
286 | |||
287 | |||
288 | * Leginon/GonioModeling> Click on <inlinegraphic |
||
289 | fileref="http://emg.nysbc.org/software/leginon/images/icons/cam_ruler.png" |
||
290 | format="PNG"/> will start "Measure" the shifts of between points that are moved on x |
||
291 | axis of the goniometer. |
||
292 | |||
293 | |||
294 | * Leginon/GonioModeling> Click on <inlinegraphic |
||
295 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG" |
||
296 | /> will "Calibrate" the scale and rotation of the model on x axis of the |
||
297 | goniometer. |
||
298 | |||
299 | |||
300 | * Leginon/GonioModeling> repeat 4-7 with the axis set to y. |
||
301 | |||
302 | |||
303 | |||
304 | |||
305 | |||
306 | |||
307 | |||
308 | |||
309 | |||
310 | h2. Acquire mosaic atlas of the grid |
||
311 | |||
312 | |||
313 | |||
314 | |||
315 | |||
316 | * scope> Remove the objective apperture. |
||
317 | |||
318 | |||
319 | * Leginon/Grid Targeting> Click "Settings" to check the preset used, the size |
||
320 | and name of the atlas. |
||
321 | |||
322 | |||
323 | * Leginon/Grid Targeting> Click "Calculate Atlas" <inlinegraphic |
||
324 | fileref="http://emg.nysbc.org/software/leginon/images/icons/calculate.png" |
||
325 | format="PNG"/> |
||
326 | |||
327 | |||
328 | * Leginon/Grid Targeting> Click "Submit Targets" <inlinegraphic |
||
329 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG" |
||
330 | />to begin acquiring the atlas. |
||
331 | |||
332 | |||
333 | * Leginon/Square Targeting> Follow the atlas progress in the display. (***There |
||
334 | is no sign at the end currently other than that nothing happens for a long time and |
||
335 | that the atlas dimension is the same in other direction when it is completed.) |
||
336 | |||
337 | |||
338 | * scope> Insert the objective aperture. |
||
339 | |||
340 | |||
341 | * Leginon/Presets Manager/Send an HM preset "To Scope" several times to cycle all |
||
342 | magnifications and warm up the objective lens to minimize the image shift hysteresis |
||
343 | in switching between LM and HM modes. If the scope has been in LM mode for a long |
||
344 | time, wait for at least 30 min before final image shift refinement of needed LM preset |
||
345 | during acquisition (i.e. sq) |
||
346 | |||
347 | |||
348 | |||
349 | |||
350 | |||
351 | |||
352 | |||
353 | |||
354 | |||
355 | h2. Determine the proper exposure condition for exposure |
||
356 | presets |
||
357 | |||
358 | |||
359 | |||
360 | Exposure presets are the base presets for data collection. To keep it to low dose |
||
361 | condition such as 10 electron/Angstrum, measurement of dose is performed and the exposure |
||
362 | time is adjusted. If scope or camera parameters are changed during the session, the dose |
||
363 | value is no longer valid and should be remeasured. |
||
364 | |||
365 | |||
366 | |||
367 | |||
368 | * Leginon/Navigation/Toolbar/Stage Locations<inlinegraphic |
||
369 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
||
370 | format="PNG"/>> Send the "empty" position "To scope". |
||
371 | |||
372 | |||
373 | * Leginon/Presets Manager/Preset Selector> Select a high mag exposure (i.e. "en" |
||
374 | or "ef"), confirm that the parameters are what you desire for the experiment, and then |
||
375 | send "To Scope"<inlinegraphic |
||
376 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
377 | format="PNG"/>. |
||
378 | |||
379 | |||
380 | * scope> Expand the beam to at least cover the CCD (> medium circle mark on |
||
381 | the viewing screen) and to the size you desire. |
||
382 | |||
383 | |||
384 | * Leginon/Presets Manager/Preset Selector> If you have changed the size or |
||
385 | location of the beam or other preset scope parameters at the scope, you must retrieve |
||
386 | the preset parameter "From scope"<inlinegraphic |
||
387 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png" |
||
388 | format="PNG"/> first before checking and saving the dose value. |
||
389 | |||
390 | |||
391 | * Leginon/Presets Manager/Preset Selector> "Acquire dose image"<inlinegraphic |
||
392 | fileref="http://emg.nysbc.org/software/leginon/images/icons/acquire.png" |
||
393 | format="PNG"/> for the selected preset. |
||
394 | |||
395 | |||
396 | * Leginon/Presets Manager/Dose Image> Check the "Dose" value. |
||
397 | |||
398 | |||
399 | * Leginon/Presets Manager/Dose Image> If desired dose is known, fill in the |
||
400 | value and then click "Match". This will change exposure time to give a close match of |
||
401 | the value entered. |
||
402 | |||
403 | |||
404 | * Leginon/Presets Manager/Dose Image> If not satisfied with the value, click |
||
405 | "No", change the exposure time by editing the preset, and try again (No need to send |
||
406 | the preset "To scope" again for the repeat) |
||
407 | |||
408 | |||
409 | * If the scope can not give such illumination to satisfy dose, exposure time and |
||
410 | area requirement, alignment is required or change of gun lens or spot size is |
||
411 | needed. |
||
412 | |||
413 | |||
414 | * Leginon/Presets Manager> Copy the preset parameter to the other exposure |
||
415 | preset. |
||
416 | |||
417 | |||
418 | * Preset Selector> send the preset that has the dose measurement "To |
||
419 | Scope"<inlinegraphic |
||
420 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
421 | format="PNG"/> |
||
422 | |||
423 | |||
424 | * Preset Selector> select the preset to recieve the copy |
||
425 | |||
426 | |||
427 | * Preset Selector> retrieve the parameters "From Scope"<inlinegraphic |
||
428 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png" |
||
429 | format="PNG"/> for the selected preset. |
||
430 | |||
431 | |||
432 | * Preset Selector/Edit><inlinegraphic |
||
433 | fileref="http://emg.nysbc.org/software/leginon/images/icons/edit.png" |
||
434 | format="PNG"/> Modify defocus for the preset after the retrieval. |
||
435 | |||
436 | |||
437 | |||
438 | |||
439 | |||
440 | |||
441 | |||
442 | |||
443 | |||
444 | |||
445 | |||
446 | h2. Update bright/dark correction images for exposure |
||
447 | presets |
||
448 | |||
449 | |||
450 | |||
451 | Exposure presets are the base presets for data collection. To make sure the best |
||
452 | correction is applied to them, the correction images should be acquired prior to the |
||
453 | 3 | Alex Kadokura | experiment at session. Since no correlation is normally done on these images, only one [[Bright and Dark reference images#Correction-Channels|channel]] is needed. Normalization image might need to be |
454 | [[Bright and Dark reference images#Correction-Planmodified]] if a large chunk of region is blocked by a |
||
455 | 1 | Amber Herold | fallen debris. |
456 | |||
457 | |||
458 | |||
459 | |||
460 | * Leginon/Presets Manager> Select a dose measured high mag exposure (i.e. "en" |
||
461 | or "ef"), and then send "To Scope". |
||
462 | |||
463 | |||
464 | * Leginon/Correction/Settings> |
||
465 | |||
466 | |||
467 | * Select TEM and camera to match that will be used. |
||
468 | |||
469 | |||
470 | * Camera Configuration: The exposure preset camera configuration |
||
471 | |||
472 | |||
473 | |||
474 | |||
475 | * Leginon/Correction/Settings> Click OK to exit settings. |
||
476 | |||
477 | |||
478 | * Leginon/Correction/Toolbar> Select "Raw image " from the pull down list of |
||
479 | acquisition modes and then click on "Acquire" button next to the selector to view an |
||
480 | image that is not corrected. |
||
481 | |||
482 | |||
483 | * Leginon/Correction/Toolbar> Select "Dark reference" in the acquisition mode |
||
484 | selector and then click "Acquire" to acquire the Dark reference image for this |
||
485 | particular camera configuration. |
||
486 | |||
487 | |||
488 | * Leginon/Correction/Toolbar> Select "Bright reference" and repeat the |
||
489 | acquisition to obtain the Bright reference. |
||
490 | |||
491 | |||
492 | * Leginon/Correction/Toolbar> Select "Corrected image" and then "Acquire" to |
||
493 | view the corrected image. A corrected image should be free of artifacts and have |
||
494 | smaller standard deviation than the raw image, in general. |
||
495 | |||
496 | |||
497 | * Leginon/Correction/Toolbar> If there are regions consistently over normalizes |
||
498 | the acquired image, either select the region and use the "+" tool to "Add Region to |
||
499 | Bad Pixel List", or click on<inlinegraphic |
||
500 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png" |
||
501 | format="PNG"/>to "Add Extreme Points to Bad Pixel List" one of more times to find |
||
502 | the outliers. Right click on a shown bad pixel removes it from the list. |
||
503 | |||
504 | |||
505 | |||
506 | |||
507 | |||
508 | |||
509 | |||
510 | |||
511 | |||
512 | h2. Medium mag preset image and beam shift |
||
513 | refinement |
||
514 | |||
515 | |||
516 | |||
517 | 3 | Alex Kadokura | Preset image shift alignment can be done follow the same procedure as the section "[[MSI set-up in more details#Grid-preset-image-shift-refinement|Grid preset image shift refinement]]" but is more |
518 | 1 | Amber Herold | efficient using the automated mode as follows. |
519 | |||
520 | |||
521 | |||
522 | |||
523 | * Leginon/Presets Manager> Send one of the exposure preset as the reference |
||
524 | (e.g. en) "To Scope"<inlinegraphic |
||
525 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
526 | format="PNG"/> |
||
527 | |||
528 | |||
529 | * scope> Move the grid to a position on the "align" square, either on a |
||
530 | recognizable object or burn a good hole through ice at the center of the CCD as |
||
531 | preparation for lower mag preset image shift correction. |
||
532 | |||
533 | |||
534 | * Leginon/Presets Manager> Click on the tool "Align presets to each other" |
||
535 | <inlinegraphic |
||
536 | fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png" |
||
537 | format="PNG"/> . The tool automatically select the reference preset that was |
||
538 | selected in the Presets Manager mainpanel and choose a preset at closest magnification |
||
539 | as the starting aligning preset. |
||
540 | |||
541 | |||
542 | * Leginon/Presets Manager/Align Presets> Click "Start" to acquire the first |
||
543 | image pair. |
||
544 | |||
545 | |||
546 | * Check, adjust, and save the preset(s) to be aligned. |
||
547 | |||
548 | |||
549 | The left panel displays and navigate by "Stage Position" of the reference preset |
||
550 | while the right panel displays and navigate by "Image Shift" of the preset to be |
||
551 | aligned |
||
552 | * Leginon/Presets Manager/Align Presets> Changing Selection of "Similar |
||
553 | look across mags" or "Full CCD" mode at the top left corner of the window will |
||
554 | cause reacquisition of the image pair in the automated mode. |
||
555 | |||
556 | |||
557 | * Leginon/Presets Manager/Align Presets> If the recongnizable object can |
||
558 | be found in both panels but not at the center on the reference left panel, |
||
559 | navigate* it to the center using its "Click Tool". Wait for the move to complete |
||
560 | and both images refreshed. |
||
561 | |||
562 | |||
563 | * Leginon/Presets Manager/Align Presets> Navigate* the object on the right |
||
564 | panel to the center using its "Click Tool". The new image shift is automatically |
||
565 | saved after each move. |
||
566 | |||
567 | |||
568 | * Repeat 3 if necessary. |
||
569 | |||
570 | |||
571 | * Leginon/Presets Manager/Align Presets> Click "Continue" to save the |
||
572 | alignment to all presets of the same magnification and continue to the next |
||
573 | preset pair. |
||
574 | |||
575 | |||
576 | |||
577 | |||
578 | |||
579 | * Repeat the process if so desired. |
||
580 | |||
581 | |||
582 | |||
583 | |||
584 | |||
585 | |||
586 | |||
587 | |||
588 | |||
589 | h2. Go to MSI Operation section |
||
590 | |||
591 | |||
592 | |||
593 | The setup that requires you to be next to the microscope is completed. Go to the section |
||
594 | 4 | Alex Kadokura | on [[MSI Operation|MSI Operation]]. |
595 | 1 | Amber Herold | |
596 | |||
597 | |||
598 | |||
599 | |||
600 | h2. Hole Targeting Set-up |
||
601 | |||
602 | |||
603 | |||
604 | We can use any image that is already loaded into Hole Targeting to adjust these |
||
605 | parameters. The values in this node vary so much from grid to grid that the best way to |
||
606 | find these parameters is by going through a step-by-step trial-and-error process. To see the |
||
607 | final acquisition and focus targets, enable only the Original, acquisition, and focus |
||
608 | 4 | Alex Kadokura | display selections. Refer to Introduction Chapter on [[MSI set-up in more details |
609 | 1 | Amber Herold | |
610 | 4 | Alex Kadokura | |how the various tools behave in the control panel]]. |
611 | 1 | Amber Herold | |
612 | 4 | Alex Kadokura | |
613 | 1 | Amber Herold | The loaded image normally comes from the image acquired through "Square" node when you |
614 | submit targets on the grid atlas in "Square Targeting" node. You can also do a practice run |
||
615 | using the default image comes with Leginon. Leave the filename entry blank when you click |
||
616 | "Load" in the hole finding settings window for the step "Original". The default values in |
||
617 | the node gives an example of reasonable target finding with such default image. |
||
618 | |||
619 | |||
620 | |||
621 | |||
622 | Setting up Hole Targeting Depends on which MSI flavor you are running: |
||
623 | |||
624 | |||
625 | |||
626 | |||
627 | 4 | Alex Kadokura | * [[Hole Targeting Set-up for MSI-Edge|MSI-Edge]] uses template matching of the |
628 | 1 | Amber Herold | edge image |
629 | |||
630 | |||
631 | 4 | Alex Kadokura | * [[JAHC finder nodes set-up|MSI-T]] uses template matching of the original |
632 | 1 | Amber Herold | image |
633 | |||
634 | |||
635 | 4 | Alex Kadokura | * [[raster finder nodes set-up|MSI-Raster]] creates raster points from a given |
636 | 1 | Amber Herold | origin |
637 | |||
638 | |||
639 | 4 | Alex Kadokura | * [[Initial MSI application preference setup#Settings-that-are-the-same-as-in-other-MSI-applications-in-queuing-mode|MSI-Tomography]] does not use automatic |
640 | 1 | Amber Herold | target finding. Therefore, it does not requires set up. |
641 | |||
642 | |||
643 | |||
644 | |||
645 | |||
646 | |||
647 | |||
648 | |||
649 | |||
650 | h2. Exposure Targeting Set-up |
||
651 | |||
652 | |||
653 | |||
654 | Like hole targeting, we can use any image that is already loaded into Exposure Targeting |
||
655 | to adjust these parameters. |
||
656 | |||
657 | Setting up Exposure Targeting Depends on which MSI flavor you are running. |
||
658 | |||
659 | |||
660 | 4 | Alex Kadokura | * [[Hole Targeting Set-up for MSI-Edge|MSI-Edge]] uses template matching of the |
661 | edge image |
||
662 | 1 | Amber Herold | |
663 | |||
664 | 4 | Alex Kadokura | * [[JAHC finder nodes set-up|MSI-T]] uses template matching of the original |
665 | 1 | Amber Herold | image |
666 | |||
667 | |||
668 | 4 | Alex Kadokura | * [[raster finder nodes set-up|MSI-Raster]] creates raster points from a given |
669 | 1 | Amber Herold | origin |
670 | |||
671 | |||
672 | 4 | Alex Kadokura | * [[Initial MSI application preference setup#Settings-that-are-the-same-as-in-other-MSI-applications-in-queuing-mode|MSI-Tomography]] does not use automatic |
673 | target finding. Therefore, it does not requires set up. |
||
674 | 1 | Amber Herold | |
675 | |||
676 | |||
677 | |||
678 | |||
679 | |||
680 | h2. Autofocus Test |
||
681 | |||
682 | |||
683 | |||
684 | There are two focusers in MSI. "Z Focus" node usually moves stage in Z direction to |
||
685 | Eucentric height at lower mags. "Focus" node usually just correct the defocus in the way |
||
686 | equivalent to turning the focus adjustment knob on the scope at a higher mag. The defocus |
||
687 | measurments in both nodes are based on the same principle of beam-tilt induced image shift. |
||
688 | A correlation (normally phase) is used to obtain the image shift. Therefore, it is important |
||
689 | to test and preform autofocus measurement at area with recognizable feature that does not |
||
690 | move significantly during the defocus measurement. |
||
691 | |||
692 | |||
693 | The test use "Manual_after" focus step in the focus sequence for checking. Two |
||
694 | procedures can be used to reach a area for good correlation measurements: |
||
695 | |||
696 | |||
697 | |||
698 | |||
699 | * Using Simulate Target tool |
||
700 | |||
701 | |||
702 | |||
703 | |||
704 | * Leginon/Presets Manager> Send the autofocus preset "To Scope" |
||
705 | |||
706 | |||
707 | * scope> Move to an area that has recognizable feature on the carbon |
||
708 | support at the center. |
||
709 | |||
710 | |||
711 | * Leginon/(Z) Focus/Focus Sequence<inlinegraphic |
||
712 | fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png" |
||
713 | format="PNG"/>> Enable desired autofocus and "Manual_after" focus |
||
714 | steps. |
||
715 | |||
716 | |||
717 | * Leginon/(Z) Focus/Toolbar> Click Simulate Target<inlinegraphic |
||
718 | fileref="http://emg.nysbc.org/software/leginon/images/icons/simulatetarget.png" |
||
719 | format="PNG"/> and follow the autofocus |
||
720 | |||
721 | |||
722 | |||
723 | |||
724 | |||
725 | |||
726 | * Submiting focus target from Hole Targeting |
||
727 | |||
728 | |||
729 | *To use this procedure, there has to be a grid mosaic, and the Hole Targeting |
||
730 | should allow user verification of targets |
||
731 | |||
732 | |||
733 | * Leginon/(Z) Focus/Focus Sequence<inlinegraphic |
||
734 | fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png" |
||
735 | format="PNG"/>> Enable desired autofocus and "Manual_after" focus |
||
736 | steps. |
||
737 | |||
738 | |||
739 | * Leginon/Square Targeting> Select a test square and submit it<inlinegraphic |
||
740 | fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" |
||
741 | format="PNG"/> |
||
742 | |||
743 | |||
744 | * Leginon/Hole Targeting> Submit a focus target to test autofocus in Z Focus |
||
745 | node and an acquisition target for the next stage. Both targets should be in an |
||
746 | area that has recognizable feature with carbon support. |
||
747 | |||
748 | |||
749 | * Leginon/Z focus> follow the autofocus test in this node. |
||
750 | |||
751 | |||
752 | * Leginon/Exposure Targeting> Submit a focus target at a location that has |
||
753 | recognizable feature with carbon support to test autofocus in Focus node. |
||
754 | |||
755 | |||
756 | * Leginon/Focus> follow autofocus test in this node. |
||
757 | |||
758 | |||
759 | |||
760 | |||
761 | |||
762 | |||
763 | |||
764 | |||
765 | Checking the performance of autofocus by the following |
||
766 | criteria: |
||
767 | |||
768 | |||
769 | |||
770 | |||
771 | * During autofocusing |
||
772 | |||
773 | |||
774 | |||
775 | |||
776 | * Display the image and correlection peak: The observed movement of images |
||
777 | upon beam tilt should be consistent with the correlation peak and the edge of |
||
778 | the beam should not be visible in any of the beam-tilt images. |
||
779 | |||
780 | |||
781 | * Display the correlation image: The correlation peak at its correct position |
||
782 | should be significantly higher than background. |
||
783 | |||
784 | |||
785 | |||
786 | |||
787 | |||
788 | |||
789 | * During manual focusing |
||
790 | |||
791 | Manual Focusing can be activated either by enabling "Manual_before/Manual_after" |
||
792 | steps or directly when leginon is idle using (Z) Focus/Toolbar/MF. To activate manual |
||
793 | focus directly, it is necessary to send to scope the preset of the choice (normally |
||
794 | fc) first, though. |
||
795 | |||
796 | |||
797 | * MF> The FFT should show visible and isotropic Thon ring according to the |
||
798 | fc preset defocus. |
||
799 | |||
800 | |||
801 | * MF/Toolbar> Make sure the move type is "Defocus" and the set value in the |
||
802 | box is 0 m then click "Set instrument" botton<inlinegraphic |
||
803 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png" |
||
804 | format="PNG"/>. This will set the defocus to the current 0. |
||
805 | |||
806 | |||
807 | * MF> The perfect autofocus result is that the FFT indicates that the scope |
||
808 | is at Gaussian focus. |
||
809 | |||
810 | |||
811 | * For relative movement, press "+" or "-". The step size is determined in |
||
812 | MF/Toolbar/Settings> |
||
813 | |||
814 | |||
815 | * To reset defocus to zero at the new focus, press the so-named |
||
816 | button<inlinegraphic |
||
817 | fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentsetnew.png" |
||
818 | format="PNG"/>. |
||
819 | |||
820 | |||
821 | * Clicking the Stop button<inlinegraphic |
||
822 | fileref="http://emg.nysbc.org/software/leginon/images/icons/stop.png" |
||
823 | format="PNG"/> exits the maunal check. |
||
824 | 2 | Amber Herold | |
825 | |||
826 | |||
827 | ______ |
||
828 | |||
829 | [[MSI Quick-start|< MSI Quick-start]] | [[Special Operation setup|Special Operation setup >]] |
||
830 | |||
831 | ______ |