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T12 TEM Quick Guide (BQ 19 Mar 2019)

 

Start of session

  • Fill/Check N2 dewar – fill if less than 3/4 full.

  • Check Gun/Column values < 10 is OK, 6 is perfect. (Setup tab)

  • Turn high tension on (yellow = active).

  • Check specimen holder is centred (search tab, side menu, reset holder).

  • Remove holder, make sure to put pressure on the blue stage plate at all times during removal, don’t push, but hold firmly in place by resting the palm of your hand on the grey stage casing and placing your fingers around the entry port onto the blue stage plate to spread the pressure.

  • Prepare your grid in the holder you intend to use, make sure you check the O-ring for any dust particles etc BEFORE you place it into the loading station for cooling!

  • If using the room temperature holder, take care not to bend or break the clasp that holds the grid in place and be mindful with the tool since it is fragile.

  • Insert specimen holder, according to detailed protocol below. Select and return appropriate holder selection from the warning message in the User Interface and wait for red light to go out and pumping to cease, before you proceed with the proper insertion protocol!

  • Turn filament on (yellow = active). Check emission is between 3 µA and 7µA (higher emission will not increase brightness, but shorten filament life)

  • Check Gun/column vales again, don’t open Column valves unless the log value is under 10 (best 6, under 15 for cryo holder).

  • Open column valves (grey = valves open).

  • Check/Set magnification to midrange (~13500-40000)

  • If you can’t see the beam, don’t touch the trackball! Refer to troubleshooting to find beam.

  • Once beam is visible, centre and focus beam using left hand trackball and intensity dial.

  • Check condenser aperture is aligned (method below).

  • Find image feature and adjust Eucentric height with the Alpha wobbler (search tab) using z height button.

  • Press Eucentric focus.

  • Insert Objective aperture, check for alignment, use objective aperture alignment procedure if not (method below).

  • Spread beam for camera safety. Lift screen and cover viewing glass.

  • Open CCD/TV tab and use search to get an image on the monitor. Adjust intensity if necessary and find instrument focus, using Live FFT to spread Thon rings to even illumination of screen. Use Preview and Live FFT for more accurate focus, size of first ring to be roughly around 1/3 of the width of the image. Use Focus and Intensity to obtain best contrast. Use Acquire to capture an image.

  • Save image in your folder in C drive.

 

Changing grids

 

  • Reset holder!

 

  • Close column valves.

 

  • Turn off filament.

 

  • Remove specimen holder.

 

  • Change grids carefully.

 

  • Replace specimen holder, according to relevant procedure.

 

  • Turn on filament. Wait for filament to heat up completely.

 

  • Open column valves if vacuum log is under 10.

 

 

End of session

 

  • Close column valves.

 

  • Turn off filament.

 

  • Remove specimen holder.

 

  • Remove grid carefully.

 

  • Replace specimen holder.

 

 

NEVER SHUT DOWN COMPUTER OR TUI!

 

 

If you are last user of day:

 

  • All instructions on how to finish your session are attached to the wall next to the T12!

 

Pay particular attention to:

 

  • Turn off filament

  • Turn off HT.

  • Activate cryo-cycle (set-up tab, side menu, cryo cycle, yellow = activated).

  • Remove N2 dewar and empty any N2 into a safe area/Styrofoam box. Place the dewar onto its side on the equipment table to allow air to circulate and any moisture to evaporate safely. Place absorbent paper towel under the anti contaminator coils to absorb any moisture condensing on the coils.

 

Abbreviations:

RP/LP - Right/Left hand panel

MF - Multi function dials

 

 NOTE! After transfer, your data should be located in:

/raid/em/t12data

Tabbing the em directory will not work due to permissions and thus you will need to enter in the entire path above to access.

Removing specimen holder, changing grids & replacing holder

 

 

To remove specimen holder:

 

Close column valves and centre specimen holder. Turn filament off.

 

Place fingers of one hand onto purple stage plate to stabilise stage whilst removing specimen holder.

 

Holding the black knob on the specimen holder, pull straight out until you feel resistance. Without releasing specimen holder turn clockwise until you feel resistance. The specimen holder is now in a safe ‘parking’ position.

 

Place fingers of left hand on purple stage plate for stability. Use the thumb/fingers of one hand to push and the other hand to pull, to guide the specimen holder gently out of the airlock (pushing with one hand/finger and pulling with the other helps to release the o-ring from the tight fit of the airlock gently without a sudden release which might lead to damage to holder or airlock.)

 

Place specimen holder in grid transfer station on the table.

 

 

To change grids:

 

Remove the end cap of the transfer station and lift the clasp with the provided tool. Place grid into the holder and close the clasp gently. PLEASE BE VERY CAREFULLY WHEN OPENING AND CLOSING THE CLASP. It is very easy to bend the clasp or break the lifting tool. Please make sure when you are doing this that the clasp is straight and not bent or twisted to either side. Make sure the clamp is in correct orientation before lowering onto grid and that the grid is securely held.

 

 

 

To replace specimen holder:

 

Line up the brass pin on the specimen holder in approximately the 5 o’clock position, matching the airlock location in the stage.

Holding the stage plate for safety, guide the specimen holder carefully into the stage (taking care not to catch the brass pin) until you feel resistance (approximately 3/4 of the length of specimen holder). The ODP Pump will automatically start and the red light on side of stage will be lit. WHEN THE RED LIGHT IS ON THE SPECIMEN HOLDER MUST NOT BE INSERTED ANY FURTHER. Please make sure you return the message on the User Interface asking for holder specification! Make sure you choose the correct holder settings for the type you are using and return the message!

 

When the pump has stopped and the red light is out, turn the specimen holder and carefully guide the specimen holder into the microscope, making sure the stage plate is supported and you do NOT let go of the holder when the vacuum pulls it inside. It is a strong vacuum, and the force of the holder being sucked into the stage can cause damage inside.

 

Aligning Condenser Aperture

 

Set magnification to mid range (~13500-40000)

 

The beam should be round when you focus through intensity (intensity button on LP). To align the condenser aperture, focus the beam, centre the beam using LP trackball and adjust the condenser x and y axes (outside screws on the condenser at the microscope) while going through crossover and checking the beam is round on either side of crossover.

 

Adjusting Z Height

 

Find a recognisable feature central to a grid, and press the “Alpha wobbler” (search tab flapout).

Adjust the movement of the feature on the screen to minimal movement using the z-Axis up and down buttons (RP).

Stop the Alpha Wobbler, when you are satisfied that movement is minimal.

Press the Eucentric focus button (RP).

This procedure has to be repeated for every new grid, as they can vary slightly in thickness.

Aligning objective aperture

 

Before inserting the objective aperture move to an area of carbon film with a clearly visible/high contrast particle to the centre of the viewing screen. Focus and centre the beam.

Press Diffraction button (RP) and check the camera length is around 660mm (D). If necessary, use intensity to spread the beam to create a focused image of the diffraction rings.

Centre the central spot using Multifunction dials X/Y.

Insert the objective aperture, check position of the aperture image on the screen and adjust around the central spot by adjusting the X and Y axes alignment screws in the objective aperture assembly.

Return to image mode by press the Diffraction button again.

 

Trouble shooting

 

No beam

  • Check column valves are open.

  • On Cryo: check shutter is open.

  • Check intensity (100% is fully spread, not condensed)

  • Lower magnification.

  • Check you are not on a grid bar.

  • Import latest alignments (alignments tab, alignments flap-out, files, choose the last by date and kV)

 

Poor focus

  • Increase intensity.

  • Set Z height.

  • Lower focus step to get finer adjustment.

 

Intensity dim

  • Import alignments

  • Check coarse button above intensity button is illuminated red.

  • Check emissions. Should be between 3µA and 7µA max. Report if emissions are less than 1µA or if any shadows are visible in the beam without a sample.

 

Column Pressure too high

  • Check liquid nitrogen is full

  • Do not open column valves if Gun/Column value is above 10.