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Document Title:
Remote Tomo5 operation guide for eBIC microscopes

Author(s):
Kyle Morris
Julika Radecke
Andrew Howe

Version:
2.0.3
Date:
27 Jun 2022

If you have any questions at any point during set-up, please ask your Local Contact for assistance Page 21 of 29

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Ensure the tracking/focus area is (mostly) on carbon with tracking features; avoid

cracks, ice contamination or overly thick regions.

Inspect the Overview and ensure the tracking/focus area does not expose a later acquisition

area

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Summary of Contents for Remote Tomo5

Page 1: ...Remote Tomo5 operation guide for eBIC microscopes Revision 2 0 3 Revision date 27 Jun 2022 Author s Kyle Morris Julika Radecke Andrew Howe ...

Page 2: ...the minimum number of autogrid boxes We standardly load grids from autogrid boxes in the number order they are present and follow the numbering written on the boxes The local contact LC will load the microscope and take an inventory of the grids in the autoloader before handover Ahead of your session please inform your local contact LC of your Target pixel size and target total dose Grid type UAu ...

Page 3: ...calibrations 15 3 2 4 Calibrate the Image Shifts 16 4 Tomography setup 17 4 1 Setup 17 4 1 1 Session setup 17 4 1 2 Identify tomography targets 18 4 1 2 1 Overview identify square or lamella ______________________________________________ 18 4 1 2 2 Lamella tilt correction if applicable _______________________________________________ 18 4 1 2 3 Search Maps medium mag montaging identify hole or cell...

Page 4: ... type mesh size 4 Grids are clipped y n 5 Number of grids 6 Any loading instructions if required 1 2 Remote connection Prior to your session please check you can log in on NoMachine as described in the Remote access and analysis guide You will not see an instrument until your session is scheduled but you should check you can successfully log in to nx cloud diamond ac uk via NoMachine 1 3 Quick sta...

Page 5: ...etween the virtual monitor hosting the TEM user interface and TFS Tomo use the Team Viewer Monitor buttons TEM Control Pads Simulator user interface The Control Pads Simulator provides access to the microscope controls you would find at the instrument itself It is in general becoming less necessary to use these controls but for some operations later in the guide it is useful to be aware of where t...

Page 6: ... Version 2 0 3 Date 27 Jun 2022 If you have any questions at any point during set up please ask your Local Contact for assistance Page 6 of 29 TEM User Interface TUI user interface It is in general becoming less necessary to use TUI but for some operations later in the guide you will need to access this interface ...

Page 7: ...sistance Page 7 of 29 Thermo Fisher Scientific Tomo software user interface You can generally understand navigation through the tomography workflow via working left to right through the Workflow tabs and top to bottom through the Workflow tasks operations For example your imaging conditions Presets are first configured under the Acquisition and Optics Settings This guide presents a suggested seque...

Page 8: ...art a new session for taking an Atlas Atlas New Session Session Setup Naming convention Supervisor_YYYYMMDD_XXXXXX_visit ID_Atlas o Do not edit Supervisor_YYYYMMDD_XXXXXX prefix created by Tomo o We strongly suggest adding the visit ID and Atlas suffix Image format MRC o These are the Atlas and Atlas tile image formats o Using MRC is recommended to retain the extended MRC header information Use th...

Page 9: ...e chosen grid s Atlas Screening Select the checkbox of each grid you want to acquire an Atlas of and select Close Col Valves If acquiring more than one Atlas go to autoloader and switch the turbo to Always On o Wait for the Turbo to reach 100 o Check Turbo status on the microscope PC TEM Life Science Autoloader Vacuum control Switch turbo to Auto Off once you have finished taking Atlas maps of you...

Page 10: ...n tab Acquisition and Optics Settings you may select the Preset parameters and adjust to suit your experiment Check your low magnification Presets using your loaded grid Take a Preview using the Atlas Preset If necessary re centre on the square Take a Preview using the Overview Preset If necessary re centre on the square and reacquire the Preview Adjust the magnification to fill the field of view ...

Page 11: ...er high mag Presets Select the Exposure Preset from the drop down Press Set to send Exposure parameters to the microscope Select each high magnification Preset Tracking Focus Drift Zero Loss Thon Ring Press Get to retrieve the Exposure parameters from the microscope and copy into the currently selected high magnification Preset Repeat for each high magnification Preset Adjust exposure times especi...

Page 12: ...e online dose calculators helpful Example exposure time calculation with parallel illumination targeting optimal dose per tilt Example exposure time calculation with parallel illumination targeting total dose Physical pixel dose rate 5 281 e px sec Pixel size magnified 1 63 Å px Pixel area magnified 2 66 Å2 px 𝒆 𝒑𝒙 𝒔𝒆𝒄 Å𝟐 𝒑𝒙 𝒆 Å𝟐 𝒔𝒆𝒄 Dose per tilt target 3 2 e Å2 Dose rate 1 988 e Å2 sec 𝒆 Å𝟐 𝒆 Å𝟐...

Page 13: ...r grid square size Find eucentric height by using the auto eucentric by stage tilt under Auto Functions Auto Functions Auto eucentric by stage tilt Presets Eucentric Height Start 3 2 2 1 Automatic eucentric height troubleshooting If Tomo fails to automatically find eucentric height there are a few things to try A courser search can help Re run Auto eucentric by stage tilt with Presets Overview for...

Page 14: ...se the Image Filter Settings for i e Low SA euc height overview tracking High SA Focus Tracking With stage set to 0 Acquire Image 1 With stage set to 5 Acquire Image 2 Click compare Look for reliably found cross correlation peak In the case of not finding a cross correlation peak try Longest wavelength nm of the scale bar Shortest wavelength nm 1 40 of the scale bar Systematically try using differ...

Page 15: ...ew using the Overview Preset Preparation Preset Overview Preview 2 Check the feature remains centred right click Move stage here if necessary 3 Collect a preview using the Search Preset Preparation Preset Search Preview 4 Check the feature remains centred right click Move stage here if necessary 5 Collect a preview using the Exposure Preset Preparation Preset Exposure Preview 6 Check the feature r...

Page 16: ...the image shift between the Presets At Exposure magnification the stage will be shifted for subsequent magnifications Image Shifts will be recalibrated Click Proceed to move to the next pair of Presets If you found the feature remained centred during targeting then you may choose to skip image shift calibrations If you needed to recentre the feature during targeting you should calibrate the Image ...

Page 17: ...ssion Session setup Naming convention Supervisor_YYYYMMDD_XXXXXX_visit ID_autoloader _Tomo o Do not edit the Supervisor_YYYYMMDD_XXXXXX prefix created by Tomo o We strongly suggest adding the visit ID autoloader position and Tomo as a suffix Sample type Slab like Options Select Batch Low Dose Dose fraction output format Tiff Lzw Non Gain normalized o These are the raw movie stacks from the detecto...

Page 18: ...phy Auto Eucentric Tomography Acquire Overview 4 1 2 2 Lamella tilt correction if applicable If working with lamella before acquiring Search images to identify targets the specimen tilt due to milling angle will need to be corrected The milling angle is known from the FIB SEM experiment at eBIC this is typically 13 however the loading orientation of the lamella is not known The schematic below sho...

Page 19: ...a If the lamella appearance change is not obvious try tilting to a higher angle i e 40 Apply the appropriate Tilt Correction by using Set Tilt and Set Acquire a new Overview with the Tilt Correction applied 4 1 2 3 Search Maps medium mag montaging identify hole or cellular targets Inspect the Overview move to a region of interest To inspect a large area at Search magnification acquire a Search Map...

Page 20: ...llection described below Reposition if required Right click Move Stage Here and Acquire Image Repeat if region of interest is not centred Enter Name for desired tomogram naming convention Enter Defocus to set desired defocus value for each tomogram Set Tilt 0 00 to define starting angle of tomogram Can be any other value between 60 and 60 4 1 3 Acquisition setup 4 1 3 1 Set up batch position Adjus...

Page 21: ...022 If you have any questions at any point during set up please ask your Local Contact for assistance Page 21 of 29 Ensure the tracking focus area is mostly on carbon with tracking features avoid cracks ice contamination or overly thick regions Inspect the Overview and ensure the tracking focus area does not expose a later acquisition area ...

Page 22: ...n Tilt the stage using Set Tilt to check tilted acquisition will be successful on the batch areas Be aware of what is close and what is going to come into the beam Look for features that will interfere with tracking and or acquisition including grid bars 4 1 3 3 Add batch position When you have validated the position is ready for collection click Add position Repeat for new targets Return to secti...

Page 23: ...un eucentric refinement before each tomogram is acquired during the data acquisition To check the eucentric height is properly calibrated for your batch positions there are a number of available strategies You may wish to choose one based on the type of tomography session you are performing single particle like cellular lamella For single particle like specimens your grid height is most likely to ...

Page 24: ...ght refer to 3 2 2 and the objective aperture should be removed Use the Auto Functions in the order as below The order should be followed if Auto Function is successful then move on to the next Ensure that you select the corresponding Preset for each Auto Function alignment 1 Auto Functions Auto Functions TEM Auto eucentric by stage tilt Start Presets Eucentric Height for Auto eucentric by stage t...

Page 25: ...0 µm objective aperture Check the beam centring Direct alignments Beam shift Multifunction X Y Repeat the Autostigmate Auto Function on a carbon area Auto Functions Auto Functions TEM Autostigmate Start Presets Thon Ring for Autostigmate 5 1 2 Zero loss peak Check the ZLP Double check you are on carbon to check ZLP centring is successful with the current Preset parameters set in 3 1 4 Note whether...

Page 26: ...t been calibrated using the AutoFunctions Please select Close Column Valves You may wish to select Skip Eucentric if you have refined positions or your grid is flat You may wish to select Adjust Exposure Time if you want to increase your dose at higher tilts You may also select specific positions for collection You may also Update Defocus to batch modify the defocus series of a set of batch positi...

Page 27: ... Tomography Tasks Data Acquisition Hit start to commence data collection Monitor the beginning of data acquisition and frequently during your session As batch positions have their tomograms collected their status will update Inform your local contact that you have started your data acquisition 5 2 3 Plan to request your dewar return Communicate with your local contact your plan for the session and...

Page 28: ... to do this Please note that Tomo will pause data collection when LN2 is being refilled or if you use a K3 camera when GIF ZLP is being adjusted However this should not last longer that 15 20 minutes Additional time might be required for Tomo to move to a new target and refine the new tomogram position Therefore if after 30 minutes no new files appear as described in the Data_access_and_analysis_g...

Page 29: ...29 5 2 5 Final steps Click on 1 the highlighted cross to close TeamViewer Take care that you do not accidentally close the TEM User Interface Click on 2 and 3 to sign out of the remote support PC Take care that you do not accidentally log out the Microscope operating system Press Ctrl Alt 0 win or Ctrl Option 0 apple on your keyboard to access NX options Click on 4 Connection Click on 5 Disconnect...

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