<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">This is great! Thank you very very much!<div class="">Actually the use of target counts is much more appropriate and efficient than a simple exposure time extension, great idea.<br class=""><div class="">I’ll give it a try as soon as I can and ask for further info if needed.</div><div class="">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.</div><div class="">Thanks a lot again!</div><div class="">Best,</div><div class=""><br class=""></div><div class="">Davide<br class=""><div class="">
_________________________________________________<br class=""><br class="">Davide Floris<br class="">PhD student<br class="">Max Planck Institute of Biophysics<br class="">Structural Biology Department<br class="">Max-von-Laue Straße 3<br class="">60438 Frankfurt am Main<br class="">Germany<br class=""><br class="">(L2.090): +49-69-6303-3037<br class="">(B2.205): +49-69-6303-3049
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<br class=""><div><blockquote type="cite" class=""><div class="">On 22 May 2017, at 11:27, Ben Engel <<a href="mailto:bdengel@gmail.com" class="">bdengel@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class="">Hi Davide,<div class=""><br class=""></div><div class="">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. </div><div class=""><br class=""></div><div class="">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.</div><div class=""><br class=""></div><div class="">I am attaching the script, and also notes from Philipp Stawski, who did most of the work.</div><div class=""><br class=""></div><div class="">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.</div><div class=""><br class=""></div><div class="">Good luck,</div><div class="">Ben</div><div class=""><br class=""></div><div class="">------</div><div class=""><br class=""></div><div class=""><b style="font-size:12.8px" class="">Hi Everyone, </b><span style="font-size:12.8px" class=""></span><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">as promised here is a version of the Hagen tilt scheme that I modified so that it can be started at any starting angle.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">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,</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">and this results in virtually the same number of counts when tilting +xº and -xº from your actual zero.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><u class="">The procedure to use the script is as follows:</u></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">0) Set up your SerialEM session as usual.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">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)</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">2) Check and adjust AngleOffset (your starting angle), CountTarget (the number of counts you’re asking for each image), step (the angle increment) </div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> 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’)</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> Make sure you’re not exceeding the stage limits!!!</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">3) Do rough and fine eucentric height.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">4) Tilt to your actual zero (i.e. ~11º for 18º milling angle).</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">5) Select record and focus area and run autofocus.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">6) Run the script from the dop-down menu.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">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).</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">8) When done, the script will tilt back to your actual zero and the log will read ‘Tomogram done!'</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">A few remarks:</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">• 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.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> 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!’).</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">• If you run another script or change a script while paused, <u class="">you will not be able to resume</u>! There is currently no option to continue an already started </div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> tomogram acquisition, but I’m working on that and will let you know once it is done.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">• When setting up your tomogram, adjust your record exposure so that you’re roughly getting the set number of counts. Otherwise your first image </div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> will be off in counts (under/overexposed).</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">• 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</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""> a 1/cos approach for increasing the exposure timing.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">If you have any questions on how to run the script or on our experience with it so far, don’t hesitate to ask.</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">Best,</div><div style="font-size:12.8px" class=""><br class=""></div><div style="font-size:12.8px" class="">Philipp.</div></div><div class=""><br class=""></div><div class="gmail_extra"><br class=""><div class="gmail_quote">On Mon, May 22, 2017 at 9:34 AM, Davide Floris <span dir="ltr" class=""><<a href="mailto:dafloris@biophys.mpg.de" target="_blank" class="">dafloris@biophys.mpg.de</a>></span> wrote:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word" class=""><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">Dear all,</div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">I’m wondering if any of you involved in electron cryo tomography has implemented the dose-symmetric tilt scheme from </div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">“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) </div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">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.</div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">Has anybody tried to modify the SerialEM macro in this sense?</div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">Thanks a lot for the kind attention.</div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">Best regards,</div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69);min-height:14px" class=""><br class=""></div><div style="margin:0px;line-height:normal;font-family:Helvetica;color:rgb(69,69,69)" class="">Davide Floris</div><div class="">
______________________________<wbr class="">___________________<br class=""><br class="">Davide Floris<br class="">PhD student<br class="">Max Planck Institute of Biophysics<br class="">Structural Biology Department<br class="">Max-von-Laue Straße 3<br class="">60438 Frankfurt am Main<br class="">Germany<br class=""><br class="">(L2.090): <a href="tel:+49%2069%2063033037" value="+496963033037" target="_blank" class="">+49-69-6303-3037</a><br class="">(B2.205): <a href="tel:+49%2069%2063033049" value="+496963033049" target="_blank" class="">+49-69-6303-3049</a>
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