Project

General

Profile

MSI set up in more details » History » Version 2

Amber Herold, 04/27/2010 11:41 AM

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
*  Initial alignment at the microscope See Chapter on <link
15
linkend="microscope_setup">Microscope setup</link> for details.
16
17
18
19
20
*  scope> Insert specimen holder
21
22
23
*  scope> Put stage to eucenter and reset defocus in both HM mode AND LM
24
mode
25
26
27
*  scope> Check gun tilt and shift
28
29
30
*  scope> Check rotation center and beam tilt pivot points in the HM
31
mode
32
33
34
*  scope> Save the goniomenter position for the square with intact
35
supporting film where the alignment was done and do the same for a square where
36
the supporting film is broken (where bright/dark images can be acquired).
37
38
39
40
41
42
43
* Start python-based leginon main program amd leginon
44
client. See Chapter on <link linkend="microscope_setup">Start Leginon</link>.
45
46
47
48
49
*  scope> Set up <link linkend="CCDnote">CCD camera</link>
50
51
52
* <link linkend="test_start_client">scope> Start Leginon Client</link>
53
54
55
*  scope> If the error message
56
57
<blockquote>
58
59
_"Exception EAccessViolation in module adaExp.exe at
60
...._
61
62
63
_Access violation at address xxxx in module 'adaExp.exe'. Read of
64
address xxxx"_
65
66
</blockquote>
67
appears, DO NOT touch it, or Leginon Client will need restarting
68
69
70
* <link linkend="test_start_main">main> start-leginon.py</link>
71
72
73
*  Leginon Setup Wizard> Create new or Select old session, connect to the
74
instrument host, and start.
75
76
77
78
79
80
81
*  Start your preferred "MSI" application.
82
83
84
85
86
*  Leginon/Application/Run> Select the "MSI" application you would like to
87
use (Edge,T,Raster, or Tomography), the scope, and the main launcher. Click
88
Run.
89
90
91
*  Leginon/Node Selector> Select "Presets Manager" node.
92
93
94
* Leginon/Presets Manager/Preset Selector> Import
95
presets from a previous working session <inlinegraphic
96
fileref="http://emg.nysbc.org/software/leginon/images/icons/import.png"
97
format="PNG"/>
98
99
100
* Select the TEM and camera to "Find" sessions closest to the condition of
101
your data collection.
102
103
104
* Use shift-key to select a series of presets if needed. Hold-down to
105
ctrl-key allows individual selection/deselection.
106
107
108
* Click "Import" button to import, the presets should appear in the
109
Presets Manager/Preset Selector window.
110
111
112
* Click "OK" to exit "Import Presets" window.
113
114
115
116
* Creating new preset and adjusting the preset are covered in Calibration
117
Chapter under <link linkend="presets_create">Creating presets the first
118
time</link>.
119
120
121
*  The cycle order should match the sequence of data acquisition to minimize
122
hysterisis. If the presets are imported from previous session, the cycle order
123
is imported automatically. As an example, the cycle for naming scheme used here
124
at NRAMM is gr -> sq -> hl -> fa -> fc -> en -> ef.
125
126
127
* Leginon/Presets Manager> Select a preset in
128
the list box and then click "To Scope" <inlinegraphic
129
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
130
format="PNG"/> to apply the settings to the scope and synchronize Leginon and
131
the scope settings.
132
133
134
135
136
137
138
* Save the alignment and empty position in Leginon
139
140
141
142
143
*  scope> Recall the stored intact or empty position or go to such
144
location.
145
146
147
*  Leginon/Navigation/Toolbar/Stage Locations>
148
<inlinegraphic
149
fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
150
format="PNG"/> Enter a New name to store Stage Location such as, for example,
151
"empty" and "align", respectively. Save the xy coordinate only. To recall the
152
position, select the named location under the same tool and click on "send to
153
scope".
154
155
156
157
158
159
160
161
162
163
164
165
166
167
h2. Grid preset image shift refinement
168
169
170
171
172
173
*  Leginon/Presets Manager> Send a preset at an intermediate mag at HM mode as
174
the reference (e.g. hl) "To Scope"<inlinegraphic
175
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
176
format="PNG"/>
177
178
179
(Note: sq preset in LM mode can be used if the image alignment between LM and HM
180
is known to be good).
181
182
183
*  scope> Move the grid to a position on the "align" square, either on a
184
recognizable object or burn a good hole through ice at the center of the CCD as
185
preparation for lower mag preset image shift correction.
186
187
188
*  Leginon/Presets Manager> Click on the tool "Align presets to each other"
189
<inlinegraphic
190
fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
191
format="PNG"/> .
192
193
194
*  Check, adjust, and save the preset(s) to be aligned.
195
196
197
The left panel displays and navigate by "Stage Position" of the reference preset
198
while the right panel displays and navigate by "Image Shift" of the preset to be
199
aligned
200
*  Leginon/Presets Manager/Align Presets> Choose the reference (hl) preset
201
on the left panel.
202
203
204
*  Leginon/Presets Manager/Align Presets> Choose the aligning (gr) preset
205
on the right panel.
206
207
208
*  Leginon/Presets Manager/Align Presets> Select "Similar look across mags"
209
mode at the top left corner of the window. This will use a smaller part of the
210
CCD to acquire the image for the lower mag preset so that it appears to be
211
magnified in the 512x512 image panel.
212
213
214
*  Leginon/Presets Manager/Align Presets> "Start" image acquisition.
215
216
217
*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
218
not be found in both panels, go back to step one to choose a lower mag preset or
219
a better object.
220
221
222
*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
223
be found in both panels but not at the center on the reference left panel,
224
navigate* it to the center using its "Click Tool". Wait for the move to complete
225
and both images refreshed.
226
227
228
*  Leginon/Presets Manager/Align Presets> Navigate* the object on the right
229
panel to the center using its "Click Tool".The new image shift is automatically
230
saved at each move.
231
232
233
*  Repeat 7 if necessary.
234
235
236
237
238
239
240
241
242
243
* "Navigate" means selecting the navigate tool<inlinegraphic
244
fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png" format="PNG"/> on
245
the top right of image display and then double clicking the location of the new center that
246
is translated by the given TEM Parameter.
247
248
249
250
251
252
h2. Grid preset stage postion refinement
253
254
255
256
Stage position model Scale and Rotation is stable as long as the LM eucentric height
257
defocus is set to zero before each session, although variation from day to day is normal.
258
Check the stage movement calibration of gr preset by navigating it in Navigation node.
259
Within 10% error is satisfactory since reaching a square does not require high accuracy. If
260
not, perform stage model position _<link linkend="gonm_m_only">scale and rotation
261
calibration</link> at gr preset_ using "Calibration" application as
262
following:
263
264
265
_<link linkend="gonmodel_how">Why goniometer model is
266
better</link>._
267
268
269
270
271
*  scope> Remove the objective apperture.
272
273
274
*  Leginon> Run "Calibrations" Application
275
276
277
*  Leginon/Presets Manager> Send "gr" preset to scope.
278
279
280
*  Leginon/GonioModeling/Settings/Measurement> Set axis=x,
281
Points=5,Tolerance=25%, and Interval=1e-05 m. Default model label is the session
282
name.
283
284
285
*  Leginon/GonioModeling/Settings/Modeling> Set "Scale and Rotation Adjustment
286
Only"=Yes. Other settings will be set automatically once measurements are
287
finished.
288
289
290
*  Leginon/GonioModeling> Click on <inlinegraphic
291
fileref="http://emg.nysbc.org/software/leginon/images/icons/cam_ruler.png"
292
format="PNG"/> will start "Measure" the shifts of between points that are moved on x
293
axis of the goniometer.
294
295
296
*  Leginon/GonioModeling> Click on <inlinegraphic
297
fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
298
/> will "Calibrate" the scale and rotation of the model on x axis of the
299
goniometer.
300
301
302
*  Leginon/GonioModeling> repeat 4-7 with the axis set to y.
303
304
305
306
307
308
309
310
311
312
h2. Acquire mosaic atlas of the grid
313
314
315
316
317
318
*  scope> Remove the objective apperture.
319
320
321
*  Leginon/Grid Targeting> Click "Settings" to check the preset used, the size
322
and name of the atlas.
323
324
325
* Leginon/Grid Targeting> Click "Calculate Atlas" <inlinegraphic
326
fileref="http://emg.nysbc.org/software/leginon/images/icons/calculate.png"
327
format="PNG"/>
328
329
330
*  Leginon/Grid Targeting> Click "Submit Targets" <inlinegraphic
331
fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
332
/>to begin acquiring the atlas.
333
334
335
*  Leginon/Square Targeting> Follow the atlas progress in the display. (***There
336
is no sign at the end currently other than that nothing happens for a long time and
337
that the atlas dimension is the same in other direction when it is completed.)
338
339
340
*  scope> Insert the objective aperture.
341
342
343
*  Leginon/Presets Manager/Send an HM preset "To Scope" several times to cycle all
344
magnifications and warm up the objective lens to minimize the image shift hysteresis
345
in switching between LM and HM modes. If the scope has been in LM mode for a long
346
time, wait for at least 30 min before final image shift refinement of needed LM preset
347
during acquisition (i.e. sq)
348
349
350
351
352
353
354
355
356
357
h2. Determine the proper exposure condition for exposure
358
presets
359
360
361
362
Exposure presets are the base presets for data collection. To keep it to low dose
363
condition such as 10 electron/Angstrum, measurement of dose is performed and the exposure
364
time is adjusted. If scope or camera parameters are changed during the session, the dose
365
value is no longer valid and should be remeasured.
366
367
368
369
370
*  Leginon/Navigation/Toolbar/Stage Locations<inlinegraphic
371
fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
372
format="PNG"/>> Send the "empty" position "To scope".
373
374
375
*  Leginon/Presets Manager/Preset Selector> Select a high mag exposure (i.e. "en"
376
or "ef"), confirm that the parameters are what you desire for the experiment, and then
377
send "To Scope"<inlinegraphic
378
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
379
format="PNG"/>.
380
381
382
*  scope> Expand the beam to at least cover the CCD (> medium circle mark on
383
the viewing screen) and to the size you desire.
384
385
386
*  Leginon/Presets Manager/Preset Selector> If you have changed the size or
387
location of the beam or other preset scope parameters at the scope, you must retrieve
388
the preset parameter "From scope"<inlinegraphic
389
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
390
format="PNG"/> first before checking and saving the dose value.
391
392
393
*  Leginon/Presets Manager/Preset Selector> "Acquire dose image"<inlinegraphic
394
fileref="http://emg.nysbc.org/software/leginon/images/icons/acquire.png"
395
format="PNG"/> for the selected preset.
396
397
398
*  Leginon/Presets Manager/Dose Image> Check the "Dose" value.
399
400
401
*  Leginon/Presets Manager/Dose Image> If desired dose is known, fill in the
402
value and then click "Match". This will change exposure time to give a close match of
403
the value entered.
404
405
406
*  Leginon/Presets Manager/Dose Image> If not satisfied with the value, click
407
"No", change the exposure time by editing the preset, and try again (No need to send
408
the preset "To scope" again for the repeat)
409
410
411
*  If the scope can not give such illumination to satisfy dose, exposure time and
412
area requirement, alignment is required or change of gun lens or spot size is
413
needed.
414
415
416
*  Leginon/Presets Manager> Copy the preset parameter to the other exposure
417
preset.
418
419
420
* Preset Selector> send the preset that has the dose measurement "To
421
Scope"<inlinegraphic
422
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
423
format="PNG"/>
424
425
426
* Preset Selector> select the preset to recieve the copy
427
428
429
* Preset Selector> retrieve the parameters "From Scope"<inlinegraphic
430
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
431
format="PNG"/> for the selected preset.
432
433
434
* Preset Selector/Edit><inlinegraphic
435
fileref="http://emg.nysbc.org/software/leginon/images/icons/edit.png"
436
format="PNG"/> Modify defocus for the preset after the retrieval.
437
438
439
440
441
442
443
444
445
446
447
448
h2. Update bright/dark correction images for exposure
449
presets
450
451
452
453
Exposure presets are the base presets for data collection. To make sure the best
454
correction is applied to them, the correction images should be acquired prior to the
455
experiment at session. Since no correlation is normally done on these images, only one <link
456
linkend="Cal_cor_channel">channel</link> is needed. Normalization image might need to be
457
<link linkend="Cal_cor_region">modified</link> if a large chunk of region is blocked by a
458
fallen debris.
459
460
461
462
463
*  Leginon/Presets Manager> Select a dose measured high mag exposure (i.e. "en"
464
or "ef"), and then send "To Scope".
465
466
467
*  Leginon/Correction/Settings>
468
469
470
*  Select TEM and camera to match that will be used.
471
472
473
*  Camera Configuration: The exposure preset camera configuration
474
475
476
477
478
*  Leginon/Correction/Settings> Click OK to exit settings.
479
480
481
*  Leginon/Correction/Toolbar> Select "Raw image " from the pull down list of
482
acquisition modes and then click on "Acquire" button next to the selector to view an
483
image that is not corrected.
484
485
486
*  Leginon/Correction/Toolbar> Select "Dark reference" in the acquisition mode
487
selector and then click "Acquire" to acquire the Dark reference image for this
488
particular camera configuration.
489
490
491
*  Leginon/Correction/Toolbar> Select "Bright reference" and repeat the
492
acquisition to obtain the Bright reference.
493
494
495
*  Leginon/Correction/Toolbar> Select "Corrected image" and then "Acquire" to
496
view the corrected image. A corrected image should be free of artifacts and have
497
smaller standard deviation than the raw image, in general.
498
499
500
*  Leginon/Correction/Toolbar> If there are regions consistently over normalizes
501
the acquired image, either select the region and use the "+" tool to "Add Region to
502
Bad Pixel List", or click on<inlinegraphic
503
fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
504
format="PNG"/>to "Add Extreme Points to Bad Pixel List" one of more times to find
505
the outliers. Right click on a shown bad pixel removes it from the list.
506
507
508
509
510
511
512
513
514
515
h2. Medium mag preset image and beam shift
516
refinement
517
518
519
520
Preset image shift alignment can be done follow the same procedure as the section "<link
521
linkend="image_shift_align">Grid preset image shift refinement</link>" but is more
522
efficient using the automated mode as follows.
523
524
525
526
527
*  Leginon/Presets Manager> Send one of the exposure preset as the reference
528
(e.g. en) "To Scope"<inlinegraphic
529
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
530
format="PNG"/>
531
532
533
*  scope> Move the grid to a position on the "align" square, either on a
534
recognizable object or burn a good hole through ice at the center of the CCD as
535
preparation for lower mag preset image shift correction.
536
537
538
*  Leginon/Presets Manager> Click on the tool "Align presets to each other"
539
<inlinegraphic
540
fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
541
format="PNG"/> . The tool automatically select the reference preset that was
542
selected in the Presets Manager mainpanel and choose a preset at closest magnification
543
as the starting aligning preset.
544
545
546
*  Leginon/Presets Manager/Align Presets> Click "Start" to acquire the first
547
image pair.
548
549
550
*  Check, adjust, and save the preset(s) to be aligned.
551
552
553
The left panel displays and navigate by "Stage Position" of the reference preset
554
while the right panel displays and navigate by "Image Shift" of the preset to be
555
aligned
556
*  Leginon/Presets Manager/Align Presets> Changing Selection of "Similar
557
look across mags" or "Full CCD" mode at the top left corner of the window will
558
cause reacquisition of the image pair in the automated mode.
559
560
561
*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
562
be found in both panels but not at the center on the reference left panel,
563
navigate* it to the center using its "Click Tool". Wait for the move to complete
564
and both images refreshed.
565
566
567
*  Leginon/Presets Manager/Align Presets> Navigate* the object on the right
568
panel to the center using its "Click Tool". The new image shift is automatically
569
saved after each move.
570
571
572
*  Repeat 3 if necessary.
573
574
575
*  Leginon/Presets Manager/Align Presets> Click "Continue" to save the
576
alignment to all presets of the same magnification and continue to the next
577
preset pair.
578
579
580
581
582
583
*  Repeat the process if so desired.
584
585
586
587
588
589
590
591
592
593
h2. Go to MSI Operation section
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.
828 2 Amber Herold
829
830
831
______
832
833
[[MSI Quick-start|< MSI Quick-start]] | [[Special Operation setup|Special Operation setup >]]
834
835
______