[3dem] Dose-symmetric tilt scheme for electron cryo tomography

Mon May 22 13:22:56 PDT 2017

Dear Wim
Ben has summarised perfectly my reason for using the scheme. I work with thick samples, so high tilts are quite problematic. And I don’t have the possibility to produce thin lamellae. If the exposure time is gradually extended I get a good result (I see it when using Latitude), but with the symmetric tilt I will also preserve the quality of the images at low tilts. Few minutes more few less are ok with me, as far as I get what I need. 
Best,

Davide
> On 22 May 2017, at 21:59, Ben Engel <bdengel at gmail.com> wrote:
> 
> Hi Wim,
> 
> For thin specimens, I can see the logic in not increasing the dose at high tilt.  But for thicker stuff like cellular lamellae or isolated organelles, you really need the extra juice at high tilt to get a reasonable image.  And since the brilliant Hagen Scheme (patent pending) has already spent the early dose on all the low tilts, I think there is very little downside to cooking the high tilts a bit more.
> 
> Honestly, I think the only drawback to this scheme is acquisition time.  But for the perfect spot on a precious lamella, I can wait the extra 30 minutes.
> 
> Best,
> Ben
> 
> On Mon, May 22, 2017 at 9:50 PM, Wim Hagen <hagen at embl.de <mailto:hagen at embl.de>> wrote:
> Dear Davide,
> 
> May I ask what your reason is for using this tilt scheme?
> 
> In my view, changing exposure time defeats the purpose of it but the tilt scheme has other advantages that some like, e.g. 'better' tracking/focussing, easier IMOD alignment of sections and lamella without gold beads, but some of these advantages can be achieved in other easier and more time-efficient ways if one is not aiming for high-resolution subtomo results.
> 
> Best,
> 
> Wim Hagen
> EMBL Heidelberg
> 
> On May 22, 2017, at 11:56, Davide Floris <dafloris at biophys.mpg.de <mailto:dafloris at biophys.mpg.de>> wrote:
> 
>> This is great! Thank you very very much!
>> Actually the use of target counts is much more appropriate and efficient than a simple exposure time extension, great idea.
>> I’ll give it a try as soon as I can and ask for further info if needed.
>> I’m attending the IMOD/PEET workshop that will be held at the Rocky Mountain Labs this summer, if I get the chance I’ll also push to get this feature implemented in the next releases of SerialEM.
>> Thanks a lot again!
>> Best,
>> 
>> Davide
>> _________________________________________________
>> 
>> Davide Floris
>> PhD student
>> Max Planck Institute of Biophysics
>> Structural Biology Department
>> Max-von-Laue Straße 3
>> 60438 Frankfurt am Main
>> Germany
>> 
>> (L2.090): +49-69-6303-3037 <tel:+49%2069%2063033037>
>> (B2.205): +49-69-6303-3049 <tel:+49%2069%2063033049>
>>> On 22 May 2017, at 11:27, Ben Engel <bdengel at gmail.com <mailto:bdengel at gmail.com>> wrote:
>>> 
>>> Hi Davide,
>>> 
>>> Yes, we've expanded Wim's script to use target counts (but did not implement 1/cos), which will give you longer exposures at the higher tilt angles.  Our script also allows you to start the tomogram at a pre-tilt (say, 10 degrees).  This is useful for tomography of FIB lamellas, which are inclined (and thus 0 degrees isn't the least tilted projection).  You can just set this parameter to 0 degrees though. 
>>> 
>>> One improvement that is still needed is a check for the stage tilt angle before acquiring a record image.  Our stage sometimes gets stuck before reaching the desired tilt and will then take an image at this incorrect tilt.  I guess this should be fairly easy to implement.
>>> 
>>> I am attaching the script, and also notes from Philipp Stawski, who did most of the work.
>>> 
>>> Also, if you find the Hagen Scheme (TM) as useful as we do, please tell David Mastronarde.  If enough of us bug him, perhaps this feature will find its way into standard tilt-series acquisition in a future release of SerialEM.
>>> 
>>> Good luck,
>>> Ben
>>> 
>>> ------
>>> 
>>> Hi Everyone, 
>>> 
>>> as promised here is a version of the Hagen tilt scheme that I modified so that it can be started at any starting angle.
>>> 
>>> For lamellae, the pre tilt is 7 º, i.e. if you’re milling at 18º, 11º stage tilt should be your actual ‘0º’. I routinely use a starting angle of 10º,
>>> 
>>> and this results in virtually the same number of counts when tilting +xº and -xº from your actual zero.
>>> 
>>> 
>>> The procedure to use the script is as follows:
>>> 
>>> 0) Set up your SerialEM session as usual.
>>> 
>>> 1) Copy the contents of the file to an empty macro slot and change parameters as needed. (Once you save your settings the script will be there permanently)
>>> 
>>> 2) Check and adjust AngleOffset (your starting angle), CountTarget (the number of counts you’re asking for each image), step (the angle increment) 
>>> 
>>>     and tilttimes (how many times the script will do +step -> -step -> -2*step -> +2*step, e.g. for step = 2, tilt times = 15 you will get ±60º around your actual ‘zero’)
>>> 
>>>     Make sure you’re not exceeding the stage limits!!!
>>> 
>>> 3) Do rough and fine eucentric height.
>>> 
>>> 4) Tilt to your actual zero (i.e. ~11º for 18º milling angle).
>>> 
>>> 5) Select record and focus area and run autofocus.
>>> 
>>> 6) Run the script from the dop-down menu.
>>> 
>>> 7) After taking the first record, the script will ask for a name for the tomogram stack file (you can open a new file before running the script, too).
>>> 
>>> 8) When done, the script will tilt back to your actual zero and the log will read ‘Tomogram done!'
>>> 
>>> 
>>> 
>>> A few remarks:
>>> 
>>> • The script can be paused at any time by pressing ‘STOP’ and resumed by pressing ‘RESUME’. However, there is a chance that it can get stuck in a loop.
>>> 
>>>   I therefore suggest you currently don’t stop it while it is waiting to take the record (i.e. Log should not say ‘Record settling!’).
>>> 
>>> • If you run another script or change a script while paused, you will not be able to resume! There is currently no option to continue an already started 
>>> 
>>>   tomogram acquisition, but I’m working on that and will let you know once it is done.
>>> 
>>> • When setting up your tomogram, adjust your record exposure so that you’re roughly getting the set number of counts. Otherwise your first image 
>>> 
>>>   will be off in counts (under/overexposed).
>>> 
>>> • If you don’t want to use counts, just comment out the lines that say ‘SetExposureForMean $CountTarget'. We can have a look how to implement
>>> 
>>>   a 1/cos approach for increasing the exposure timing.
>>> 
>>> 
>>> If you have any questions on how to run the script or on our experience with it so far, don’t hesitate to ask.
>>> 
>>> 
>>> Best,
>>> 
>>> Philipp.
>>> 
>>> 
>>> On Mon, May 22, 2017 at 9:34 AM, Davide Floris <dafloris at biophys.mpg.de <mailto:dafloris at biophys.mpg.de>> wrote:
>>> Dear all,
>>> I’m wondering if any of you involved in electron cryo tomography has implemented the dose-symmetric tilt scheme from 
>>> “Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging” (Hagen WJ, Wan W, Briggs JA; JSB, 197(2):191-198; Feb 2017) 
>>> to use longer exposure times as the tilt angle increases. In latitude it’s possible to do this by selecting the option “1/cosine^n” in the exposure time parameters, but of course it is not possible to use a symmetric acquisition.
>>> Has anybody tried to modify the SerialEM macro in this sense?
>>> Thanks a lot for the kind attention.
>>> Best regards,
>>> 
>>> Davide Floris
>>> _________________________________________________
>>> 
>>> Davide Floris
>>> PhD student
>>> Max Planck Institute of Biophysics
>>> Structural Biology Department
>>> Max-von-Laue Straße 3
>>> 60438 Frankfurt am Main
>>> Germany
>>> 
>>> (L2.090): +49-69-6303-3037 <tel:+49%2069%2063033037>
>>> (B2.205): +49-69-6303-3049 <tel:+49%2069%2063033049>
>>> 
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