Steps to recording a Low Dose tomogram

• Zero image shifts using Tecnai module
• Align Search/Exposure in the Tecnai Low dose page (see comments below)
• Setup Serial EM lowdose modes (see comments below about dose)
  • be sure to leave yourself in Record mode
• Double check Search/Exposure Alignment
• Go to Search mode (Tecnai low dose) and locate specimen of interest
  • try to keep to center of grid and center of grid square to maximize tilt range
• Adjust eucentricity (using A wobbler in Search mode)
• Check tilt range (in Search mode)
• Go to Exposure mode (Tecnai Low dose) - you will still be in Record mode
• You should be near eucentric height, so push "Eucentric Focus" button
• Fine adjust eucentric using Serial EM (Tasks -> Eucentric fine
• Recenter sample which will have moved a bit
  • make sure you are in Record mode

• Refine centering of sample
  • give a bit of defocus so you can see something (2-8 um)
  • Take a View image (lower mag for overview) - don't adjust yet
  • Take a Preview image (lower dose for accuracy)
  • right-click-drag sample to center (this will impose an image shift)
  • Take another Preview image (to use as a reference for initial alignment)
• Write down C2 lens setting (you'll need this later)
• Tilt Series -> Setup
  • setup and record tilt series from zero to +(or -) extreme

• Scroll through the buffers and you will Trash.findDFdf an image of the initial zero tilt
• go back to zero tilt (beam blanked)
• make sure you are in Record mode
• adjust C2 lens to starting value (if necessary - depends on whether you chose to adjust intensity during the tilt series)
• Refine centering of sample
  • take View image
  • take Preview image
  • right-click-drag to center
  • Take another Preview image (for reference)
• Tilt Series -> Setup
  • you probably don't need another zero tilt image

Dose rates for low dose tomography

• Most people are using 30-60 electrons/A**2 for the entire tomographic dataset
  • Taylor: 60 images (cos rule), +/- 70deg, 43kx, 8-10um df, 30 elec/A**2
  • Agard: -60 -> +56deg, 1deg intervals, 6um df 50 elec/A**2
• 0.5 elec/um**2 on film gives 0.7 OD (exp time same as indicated on Tecnai meter)
• 14kx, 1sec reading -> 1 elec/A**2/sec
• 25kx, 1.3sec reading -> 2 elec/A**2/sec
• for 60 images, plan on 0.5-1 elec/A**2/picture
  • View: 1.3 sec reading at 25kx, 0.25 sec exposure, bin 2 -> 850 counts
  • Preview set to bin 4, 0.05 sec;
  • View (10kx) binning 4, 0.05 sec,
  • Trial bin 4, 0.1 sec
  • Focus bin 2 (or 4), 0.1 sec

Problems aligning Tecnai low dose with Serial EM lowdose

Low Dose Modes

• View is meant to be lower magnification (e.g. <10kx) with low dose, full CCD with moderately high binning (e.g. 4x). No displacement of image or beam. This setting used for eucentric height adjustment and for walk-up.
• Focus is meant to be sub-area at same mag as Record with similar binning (e.g. 2x binning with wide-quarter). Though we have also used full area with 4x binning with good success. The latter was necessary when there were no features otherwise visible. Remember that the bottom of the camera must be included in the Focus image (see "Dark band across images" below).
  • Focus area is deflected away from Record area - generally the same settings as Trial
• Trial is deflected away from Record area and used for tracking. It usually full camera at 4x binning. Generally keep deflection for Trial same as for Focus. Beam should be about the same size as the camera so that it doesn't overlap the Record area. It is ok to see the edges of the beam in Tracking and Focus.
• Record No displacement of beam or image. Set beam a bit larger than the camera. If selecting the option "Vary intensity as 1/cosine of angle", then the beam will become smaller during the tilt series and this should be taken into consideration. Also, if starting from zero and recording tilt series in 2 runs, then the C2 lens needs to be reset before starting the second run (just write down the C2 current setting at the start and reset it to that value before doing the 2nd half).
• Preview Same as Record, but with ~1/5 of the dose. Use higher binning. Will be used for an alignment step if selected in Tilt Series Setup (but not necessary for successful tomogram). Useful for final specimen centering prior to initiating the low-dose tilt series (View doesn't have the same center and Record is at higher dose).

Dark band across images

Finally I think we understand this. A dark band was occurring across the "record" images. In the first instance, this was attributed to the dark field image acquired immediately before the tomographic series. Later on, we established that it occurred with all images (we took "raw" images and didn't do dark field subtraction.

Problem is that charge remains on the camera after taking the focus image. The camera does not get cleared prior to the next exposure and the focus image only read out part of the field. One solution is to make the focus image use the whole camera. This takes longer. Another solution is to move the "wide quarter" focus area down to the bottom of the field. This forces the camera to clear all the charge. SerialEM deals with the potential problem of this focus not being on the tilt axis - even in low dose mode.

Macros language

t = track a = align f = focus r = record

Calibrations

Calibrations mostly went ok. We did experience problems with Image Shift calibrations resulting in blurred images. This was solved as follows:

This pattern of smearing suggests that image shift has already started for the next picture when the exposure was being taken. Bigger shift would start faster and smear more. So something could be wrong with my unbearably clever "Post-exposure actions" in which image shift and tilting are done during the anticipated image readout.

TO check this out, turn off "Post actions" in the Camera menu. Try the IS calibration and see if this fixes the problem. If so, then set DefaultActAfterExposures to 0 in the properties file to turn the feature off by default. Alternatively, you can increase the "ExtraBeamTime" setting and see if a reasonable increase also fixes the problem. (I would keep it under a second).

But Eyal reports that his image shift calibration problems were solved by turning off "Post actions". Lengthening the ExtraBeamTime did not help. I suggest you go ahead and set DefaultActAfterExposure to 0

Trial mode is generally used for tracking and quick binned shots and the the exposure is the basis for a number of the binned images taken in calibration routines.

Image Shift calibration explanation:

The basic calibration takes 9 pictures: centered, +X IS, centered, -X IS, centered, +Y IS, centered, -Y IS, centered. A bit of overkill but it allows the routine to subtract out drift (and slow image shift also, I guess). When you calibrate from scratch, it takes a first set with movements predicted to be 1/10 of the field size, then takes a second set with movements 1/2 of the field size. You should only have to do it "from scratch" once, and use the regular routine thereafter.

You do need a calibration for all the mags that might be used during a tilt series. Namely for your anticipated working mags as well as mags down to the lowest that will get used in any of the low mag tasks (4700x ? Check the rough eucentricity routine which has the lowest mag requirement.). That way all the movements that it makes when measuring things at lower mag will position things correctly at the working mag.

I am afraid this does not try to derive pixel size or tilt axis info. That has to be inserted from separate measurements.

limiting image shift not necessary on Tecnai F20

> The tilt series failed when it reset the image shift and tried to move the
> stage to recenter the object. I hadn't done any stage calibration, so
> this is not surprising. But the stage calibration help page talks
> about montaging and low-mag functions, so I am a little insecure about
> whether this calibration applies to resetting the image shift coils at say
> 25kx.

Right! There is a fallback stage calibration that is close to unity - although I am worried that if you tried to do a stage calibration there is a bad entry in the SerialEMcalibrations.txt file. Take a look there and if you see one snip it out.

On a 200 KV scope you can set the image shift limits large and not have to have an image shift reset during a tilt series - provided it is successfully reset at the start and the fine eucentricity works OK. But if image shift is poorly calibrated, this operation will not succeed.

low mag calibrations

Required for the Navigator module. Use slot grids with large area

Configuration

Used Weizmann configuration for Tietz F415 and Tecnai F20. Since we are using EMMenu 3.0, no further configuration was necessary.

Hi David,

Eyal told me that EMSerial is working now, the problem seems to be solved. When you use CAMC4 that comes with EMMENU4 you have to have the settings for the cameras also in the ini files of EMMENU4. It sounds a little bit complicated, but actually is very easy.

For example let's say you have the same system like in Israel, 2 cameras : Fastscan and the SCX 415. All the ini files are located in the folder C:\TVIPS\EMMENU 4.0\Config in the CAMC.ini you need the set: [CAMC] AVL_FASTSCAN=1 AVL_SCX=1

For every camera there is a Cameraxx.ini file (e.g Camera00.ini for Fastscan and Camera01.ini for the SCX). If one of this files are missing write me an e-mail.

If David Stokes's system is not cofigurated like this he will have the same problem like Eyal.

Regards, Cristian

Montage Notes

• Low mag mode - 220X (195X with screen down)
• Spot size 6 (6 s exposure time on small pad), beam completely spread
• Montage images taken in Record mode (0.2-0.4 s exposure time)
• Good idea to keep tracking on during lowest-mag montage in order to view progress
• Tilt axis is ~100-110 degrees in the onscreen ccd images
• Good idea to empty buffer tank before starting montage, otherwise it may start during acquisition

1. After setting up exposure level and magnification, take a test image. Note minumim/maximum densities. This will be used to set levels later.
2. Choose to "Align Pieces in Overview" in Montage pane. Also choose to view montage when done.
3. Open the Navigator window.
4. For subset of grid
   
   • Mark 4 points to make a large rectangle to cover most of grid
     
   • Mark each point as a Corner Point using checkbox
     
   • Navigator Menu - Setup Corner Montage
     
5. For whole grid
   
   • Navigator Menu - Setup Full Montage
     
   • Note - at 220K, binning 4, full montage is 16X14 images.
     
6. Choose "byte" rather than integer to save memory space. Bytes have range 0-255, so it asks range for scaling. Get range from values in test image.
7. Choose a reasonable filename.
8. Go over to Montage pane (left bottom of screen), click start. Be sure you are ok - you can't stop it once it is going.
9. Come back in ~30 minutes for a full grid.
10. Once done, click "New Map" in Navigator window. You should now have a montage of the whole grid.

Next steps: Trash.findDFdf interesting ares of grid for higher-mag views. However, the 2 magnifications must be aligned as follows

1. Increase magnification to 2500 or 3500X. May have to adjust brightness, spot size to keep dose low.
2. Find an interesting area of the grid, with some recognizable features.
3. Choose "Add Polygon", draw a polygon to map out this interesting area. Make it larger than 1 square, since alignment is likely far off.
4. After polygon is drawn, mark it as "Acquire" with checkbox.
5. Under Navigator menu, choose "Setup Polygon Montage". Again, choose bytes. May need another test image to estimate levels.
6. Once set up, click "Start" on Montage panel.
7. Important - After done, increase Register level from 1 to 2. Otherwise you can't do alignment.
8. Now that register has been changed, click "New Map" in Navigator Window
9. You now have 2 maps: Low-mag with Register 1, higher-mag with Register 2.
10. Compare high mag and low mag montages to Trash.findDFdf an equivalent point. The alignment may be off by 2 or 3 gridsquares.
11. To make this easier, do a screen capture of high mag image (shift-printscreen), paste into MS Paint application.
12. Change register back to 1, load low-mag map, (map 1), choose add point, and add a point on the feature.
13. Mark it as a "Registration Point" using checkbox in Navigator window.
14. Increase Registration to 2, load map 2 (high mag map), add a point on the matching feature, mark it as a registration point.
15. Go to Navigator Window, choose Transform. This will transform map 1 (low mag) onto map 2.
16. Re-load low-mag map. Note that it is now listed as Map 2, since it has been aligned.
17. Choose "Add Polygon", and draw a polygon to map out a grid square (any square)
18. Choose "Setup Polygon Montage" under Navigator menu.
19. Find areas of the grid you are interested in, and add points to the middle of these squares.
20. Mark all these points as "Acquire" using checkbox in Navigator window
21. Under Navigator Menu, choose "Acquire Areas". This will acquire polygons around all your points.
22. Once you Trash.findDFdf good areas to image, you can go to them directly using SerialEM.

Visit the Cryo EM Website at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 28 Jul 2005 -- BillRice - 02 May 2006

• Set ALLOWTOPICCHANGE = CemfacGroup