Introduction
Set up the Thermo Scientific Talos 120 TEM for automated over night acquisition. This guide helps to adjust the microscope to the specific settings needed for data, that is used to train a DNA-fork classifier algorithm.
The microscope has to be well aligned to be used in this guide.
Parts
No parts specified.
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Left screen with TEM User Interface and Flucam Viewer
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Right screen with Maps software.
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Support PC for data transfer and guide display.
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Left control panel
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Right control panel
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Talos
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Stage of the Talos
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Click in order to see the pop-up menu.
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Open the tab "Stage²" in the TEM user interface.
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Reset the Stage.
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Gently pull the holder out until you feel a resistance and turn it clockwise until you feel a limit stop.
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In this position the holder can stay, if you need to rearrange your hands.
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Pull the holder out completely.
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Open the holder clamp with the supplied needle
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Place the grid into the groove with the forceps.
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You can position the grid by gentle tapping the finger against the transparent tube of the TEM holder
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Orientate the grid as displayed. The precise angle is important for the later set up.
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Press "Turbo Pump", if the button is grey. If it is already yellow continue with the next step.
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Align the small pin of the holder with the slit in the 5 o'clock position (during next step).
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Push the holder until you feel a resistance.
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The red light is on until the evacuation of the airlock is finished. Only continue if the light disappeared.
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The pumping takes two minutes. If the countdown in the left screen is over the light is still on for a few more seconds.
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Confirm the Single Tilt specimen holder by clicking on the arrow.
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Turn the holder counter clockwise, once the red light turned off.
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The vacuum in the column should go below Log 30 before you continue working.
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Open Maps from the icon on the taskbar, if not already open.
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Generate "new project"
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Type in you project name
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Project Path should be set to "H:\"
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Press „Create“
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center view on sample
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rename layer to „overview“
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select template "117 nm"
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add new tileset
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Click "to microscope"
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The Screen must be inserted to protect the main camera at this step.
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If not inserted, insert screen.
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Open the column valves. By clicking on "Col. Valve Closed"
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The screen current must be between 5 nA and 7 nA. If this is not the case see next step.
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Use the intensity knob on the left panel.
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Turn it counter clockwise to increase screen current.
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Turn it clockwise to decrease screen current.
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Use the "fine" and "course" to adjust sensitivity of the knob.
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Press Eucentric focus on the right panel to reset the focus of the lenses.
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Press L2 (Alpha Wobbler) on the left control panel. The stage begins to tilt between +/-15°
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Reduce the movement of the image in the Flu cam Viewer (left screen)
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by changing the Z height with the Z axis button on the control panel
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sensitivity can be adjusted by + and - buttons above the joystick
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The Z-height is shown in the User Interface on the left Screen. Use it as an orientation. (Z-values are usually between -20 um and +20 um)
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The image center moves to the sides.
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The images stays centered.
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Press Wobbler on the right control panel
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Turn the upper part of the Focus knob on the right panel to stop the image movement
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The sensitivity can be adjusted by the lower part of the button and the Focus step is shown in the user interface. Smaller numbers mean more sensitivity''
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If you went to far off with the focus, press the button "Eucentric focus"
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Example, where the images first moves a lot and does not move anymore, when the sample is focussed.
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Import the values from the microscope to Maps with the button "from microscope" in the overview tileset in Maps
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Click on the arrow to open the pop up menu with the job list.
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Make sure the option "Close Column Valves when Done" is activated.
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Press "Run" in Maps
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Go to Results tab, press Show histogram
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Choose „''AUTO“'
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Navigate with the mouse in Maps
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Search for your regions of interest.
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Center view on the Region of interest
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Add a new Layer
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Rename layer to highmag
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Choose a template with the desired pixelsize from the drop down menu.
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Choose a tool from the top bar
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Create a tile set on your ROI
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Press insert screen to insert the Flu screen (left screen)
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"Screen is inserted"
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Make sure column valves are opened. Otherwise click on "Col. Valve Closed"
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Press "to microscope" in the highmag tileset
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Make sure the stage is positioned on your sample.
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Check if the screen current is exactly 5 nA
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Adjust screen current if necessary with the intensity knob on the left panel
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"Fine" and "course" adjust sensitivity of the intensity knob
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Press Eucentric focus on the right panel to reset the focus of the lenses.
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Press L2 (Alpha Wobbler) on the left control panel.
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Reduce the movement of the image in the Flu cam Viewer
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by changing the Z height with the Z axis button on the control panel
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sensitivity can be adjusted by + and - buttons above the joystick
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The Z-height is shown in the User Interface on the left Screen. Use it as an orientation. (Z-values are usually between -20 um and +20 um)
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press L2 again to stop the Alpha Wobbler
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Press R1 on the right panel to insert the Flu screen (left screen)
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Double click with the mouse, in order to move the stage (pink square in Maps) to the center of your region of interest.
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Click on "autofocus" (left screen) and wait until autofocus has finished (button turns grey again)
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Click on "from microscope", when the autofocus has finished.
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Go back to the step "Set up Acquisition Tile Sets" and repeat the steps you did for the first ROI for every ROI you want to be acquired in this project.
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Continue, if you have set up enough ROIs.
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Open the pop up menu.
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Check the job list and select only the tile sets you want to be acquired.
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Make sure the option "Close Column Valves when Done" is activated
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Press "Run"
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Stop the Maps Job, if it is still running.
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Check if column valves are closed.
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Close them, if they are open.
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Gently pull the holder out until you feel a resistance and turn it clockwise.
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In this position the holder can stay, if you need to rearrange your hands.
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Pull the holder out completely.
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