Diamond Remote Tomo5 User manual

Remote Tomo5 operation guide for eBIC microscopes
Revision: 2.0.3
Revision date: 27 Jun 2022
Author(s): Kyle Morris
Julika Radecke
Andrew Howe

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 2 of 29
Introduction
This document is intended as a guide for trained eBIC users to assist them in setting up their
data collection session at eBIC on a Krios, K3 and GIF. The guide is principally aimed at the
remote user but is applicable to on-site operation. The level of detail is targeted at a standard
eBIC operator. The user is ultimately responsible for their use of eBIC and Diamond systems. If
you are in doubt about an action you need to perform or are unable to progress, then please
reach out to the eBIC Local Contact assigned to your session for assistance. Additionally, please
report any errors in this guide to your Local Contact or [e-mail].
Please consult the following documentation for guidance on remote microscope access, data
analysis and transfer: “Remote access and analysis”
Information box colour key
User and local contact expectations
Please consolidate your grids into 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, QF, Cflat or Lacey) and number of grids
Your LC requires this information to complete energy filter tuning, camera gain preparation and
grid loading. The gain will be found in your visit processing directory. Your LC will complete
essential alignments (beam shift, pivot points, aperture centring) prior to your session,
but the user will complete Image Shift Calibrations and Auto Function alignments prior
to starting collection.
Please consult with your LC to decide on a plan if you wish to recover grids from your session.
Grids can be recovered from the cassette, but not the microscope column.
Getting prepared
Establish contact with the Local Contact for your session via yours and your LC’s preferred
choice of communication i.e. Teams, Mail, Notepad, etc.
Refer to the ‘Remote access and analysis’ guide to access the microscope via NoMachine
Information boxes are presented in
green
Optional action are presented in yellow
Lamella specific instructions are in pink

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 3 of 29
Contents
Contents ............................................................................................................................................3
1Preparation ................................................................................................................................4
1.1 Communication..................................................................................................................4
1.2 Remote connection ...........................................................................................................4
1.3 Quick start guide ...............................................................................................................4
1.4 Reasonable session timings............................................................................................4
2User interface summaries........................................................................................................5
3Microscope setup and screening ...........................................................................................8
3.1 Grid loading, Atlas and Presets.......................................................................................8
3.1.1 Initial checks and start up ..........................................................................................................................8
3.1.2 Collect your Atlas.......................................................................................................................................8
3.1.3 Inspect your Atlas.....................................................................................................................................10
3.1.4 Check your imaging condition Presets ....................................................................................................10
3.1.5 Dose calculation.......................................................................................................................................12
3.2 Image Shift Calibrations .................................................................................................13
3.2.1 Prepare for image shift calibrations .........................................................................................................13
3.2.2 Find eucentric height................................................................................................................................13
3.2.2.1 Automatic eucentric height troubleshooting _________________________________________ 13
3.2.2.2 Image Filter Settings troubleshooting ______________________________________________ 14
3.2.2.3 Manual eucentric height troubleshooting ___________________________________________ 14
3.2.3 Target a feature for Image Shift calibrations............................................................................................15
3.2.4 Calibrate the Image Shifts........................................................................................................................16
4Tomography 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 cellular targets)__________________ 19
4.1.3 Acquisition setup......................................................................................................................................20
4.1.3.1 Set up batch position___________________________________________________________ 20
4.1.3.2 Validate batch position _________________________________________________________ 22
4.1.3.3 Add batch position_____________________________________________________________ 22
4.1.4 Eucentric height refinement strategy .......................................................................................................23
4.1.4.1 Refine target positions__________________________________________________________ 23
5Final checks and data collection ..........................................................................................24
5.1 Auto Functions and alignment ......................................................................................24
5.1.1 Alignments via Autofunctions...................................................................................................................24
5.1.2 Beam centring..........................................................................................................................................25
5.1.1 Objective aperture....................................................................................................................................25
5.1.2 Zero-loss peak .........................................................................................................................................25
5.2 Start automated acquisition...........................................................................................26
5.2.1 Check your tomography acquisition parameters .....................................................................................26
5.2.2 Start tomography acquisition ...................................................................................................................27
5.2.3 Plan to request your dewar return ...........................................................................................................27
5.2.4 Monitor your data collection.....................................................................................................................28
5.2.5 Final steps................................................................................................................................................29

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 4 of 29
1 Preparation
1.1 Communication
Prior to your session, please reach out to your local contact with the following information about
your session and the grids you have shipped.
1) Target pixel size:
2) Target total dose:
3) Grid 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 start guide
1) Make your Atlas session
2) Atlas your grids
3) Check your Presets
4) Check Image Shift Calibrations
5) Make your Tomo session
6) Batch position setup
7) Position refinement
8) Perform AutoFunction alignments in EPU
9) Start data collection
1.4 Reasonable session timings
TBA

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 5 of 29
2 User interface summaries
Team Viewer
To switch between 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 to find them.
Do not put the microscope into diffraction mode
Take care not to accidentally press this button

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 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.

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 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 sequence through the tomography workflow.

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 8 of 29
3 Microscope setup and screening
3.1 Grid loading, Atlas and Presets
3.1.1 Initial checks and start up
Your Local Contact will have loaded and inventoried the autoloader cassette
Note you may find the previous sessions grid on the column or unloaded to cassette position 1
If Tomo5 is not loaded, begin by starting this software from the TEM PC
3.1.2 Collect your Atlas
Session setup:
We find it can help with set up to have a grid Atlas to navigate, so collect one first
Start a new session for taking an Atlas (Atlas > New Session > Session Setup)
-Naming convention: Supervisor_YYYYMMDD_XXXXXX_visit-ID_Atlas
oDo not edit Supervisor_YYYYMMDD_XXXXXX prefix created by Tomo
oWe strongly suggest adding the visit ID and Atlas suffix
-Image format: MRC
oThese are the Atlas and Atlas tile image formats
oUsing MRC is recommended to retain the extended MRC header information
-Use the default SSD storage folder path: H:\DATA or G:\DATA or D:\DATA
oNever use C:\
Session set-up can then be controlled by users directly through the Tomo interface. Changes to beam settings
should all be controlled directly through the Tomo Preparation presets and navigation around the grid can be done
via right-click options over the images in Preparation, Atlas and Tomo image windows.
If you are returning to this section to re-acquire an Atlas after Image Shift Calibration:
oSelect the grid(s) > Reset Selected
oSkip Session setup

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 9 of 29
Atlas collection:
Collect an Atlas of the 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”.
oWait for the Turbo to reach 100 %
oCheck 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 your grids.
-Select “Close Col. Valves” if you leave the microscope unsupervised
Click the ‘Start’ button

Document Title:
Remote Tomo5 operation guide for eBIC microscopes
Author(s):
Kyle Morris
JulikaRadecke
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 10 of 29
3.1.3 Inspect your Atlas
Inspect your single or multiple Atlases for targets by clicking on the grid in the left panel
-Left-click drag to move around, middle scroll to zoom in-out
When you have chosen your grid for target setup, select it and click ‘Load Sample’ in Tomo
3.1.4 Check your imaging condition Presets
Navigate to a square in your Atlas, right-click > Move stage here
In the Preparation 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 with the grid square
-For lamella, the Overview preset should be adjusted to fill the field of view with one
lamella
Important: Only load grids from inside Tomo, any shifts and rotations that occur to the grid during
loading and unloading will be measured and accounted for automatically. If you notice navigation on the
Atlas places you in the incorrect area this may have failed and you will need to reacquire your Atlas.
Overview - 540 X
Overview - 320
X