Spectra 300 STEM Alignment Guide (DRAFT)

This guide covers STEM alignment on the Spectra 300 at Stanford SNSF (Stanford Nano Shared Facilities). Screenshots and instructions are provided by Andrew Barnum. Written instructions and images are organized by Sangjoon Bob Lee.

Check before starting your session

First, visually confirm the following from the previous user to ensure no damage has occurred.

• Standard gold nanoparticle sample on a single-tilt holder is loaded.
• Logbook is checked for any notes from the previous user.
• Start your session on NEMO.
• Screen is inserted.
• Beam is blanked.
• Column valves are closed.
• Turbo pump is off.
• Stage tilt is at 0° (alpha and beta) and the stage has been reset.
• Arina detector is retracted.
• Arina detector is turned off.
• All holders are capped and placed in the holder box.
• No errors are found across all software programs including TEMUI.

Report immediately in the logbook if anything has occurred or contact supervisors.

After you have checked the states,

• Follow any special instructions and warnings posted on NEMO.
• Emergency contacts are available.

Acronyms:

• mulXY - Multifunction X/Y knobs on hand panel
• TEMUI - TEM User Interface (software)

Workstation layout:

Overview

This guide covers three main phases:

Part 1: Setup & Alignment

1.1 Vacuum check

Before imaging, verify that the vacuum system is ready and the column valves can be safely opened. Poor vacuum conditions can damage the electron source and contaminate the sample.

• Check vacuum status

  1. In TEMUI, open Setup tab.

  2. Locate vacuum status panel. Status shows “All Vacuum (Closed)” and Col. Valves Closed button is yellow.

     

• Verify vacuum pressure

  1. Check vacuum pressure values on the log scale (lower = better):

     Gauge	Typical Log Value	Notes
     Gun	1	Critical for source lifetime
     Liner	14	Column vacuum
     Octagon	1	Sample area
     Projection	19-21	Below sample
     Buffer tank	41-51	Empty if above 51

  2. If buffer tank pressure is above 51, click Empty Buffer.

     • Pumps cycle audibly.
     

  3. Wait for value to decrease before proceeding.

     

• Open column valves

  1. Click Col Valves Closed button.

     • Status changes to “All Vacuum (Opened)”.

     NOTE: The system only allows opening if vacuum levels are acceptable. Once opened, the electron beam path is clear from gun to sample.

     

1.1 Check convergence angle

For this guide, we use a convergence angle of 30.0 mrad.

• In TEMUI, navigate to the Tune tab, then select Aperture.
• Set Condenser 1, Condenser 2, and Condenser 3 to 2000, 70, and 1000.

NOTE: These aperture values determine the convergence angle. For example, setting Condenser 2 to 50 instead of 70 gives a convergence angle of 21.5 mrad. A smaller aperture restricts the beam to a narrower range of incident angles, blocking higher-angle electrons.

1.2 Find eucentric height

Complete eucentric height alignment after loading each sample and before imaging. Do not skip this step. At eucentric height, the sample remains stationary when tilted. This is essential for accurate imaging and tomography. The ronchigram “blow-up” method provides a quick way to find this position.

• View ronchigram

  1. Verify the Diffraction button is pressed on the hand panel with the red light turned on.

     NOTE: The ronchigram is the diffraction pattern formed when the convergent probe is stationary. When defocused, it contains shadow images of sample features, making structure visible during z-height adjustment.

  2. In TEMUI, view the ronchigram in the main display.

     

  3. Position probe on a sample region that scatters electrons (not over a hole or vacuum).

• Adjust z-axis to find blow-up point

  1. Lower magnification to 5,000x. A wider field of view makes ronchigram changes easier to observe.

  2. Find a region where there is a sharp contrast at a boundary, as shown in the following image.

     

  3. Use z-axis buttons on hand panel to move stage up or down.

     • Buttons are pressure sensitive: press harder for faster movement.
     • Start with gentle presses for fine control.
     • Notice that as you adjust the z-axis, the ROI also shifts. Use the joystick to remain on the sharp contrast boundary region.

  4. Watch the ronchigram while adjusting z-height. The pattern “zooms” in or out as the sample moves through focus.

  5. Continue adjusting. The ronchigram expands when approaching eucentric height, also referred to as the “blow-up” point.

     

  6. Find the “blow-up” point where the ronchigram appears infinitely magnified: shadow image features expand until they fill the entire display.

     • If the ronchigram starts shrinking again, reverse direction.
     

1.3 STEM mode configuration

Before performing alignments, configure the STEM imaging parameters and verify detector settings.

• Enable descan

  1. In TEMUI, locate the STEM Imaging (Expert) panel.

  2. Enable Descan checkbox.

     NOTE: Descan compensates for beam movement during scanning, keeping the diffraction pattern stationary on the detector.

     

• Verify HAADF is retracted

  1. Locate the Selection panel.

  2. Verify detector states:

     • BF-S (Bright Field): Retracted
     • DF-S (Dark Field): Retracted
     • HAADF: Retracted

  3. Toggle HAADF checkbox on then off to confirm retracted state.

     NOTE: HAADF must be retracted during ronchigram alignment. The HAADF is a ring-shaped detector with a central hole. If inserted, high-angle electrons hit the ring instead of the camera below, blocking part of the ronchigram.

     

• Set detector layout in Velox

  1. Open Velox acquisition software.

  2. Open detector layout display.

  3. Set camera length to 91 mm.

     NOTE: Camera length determines detector collection angles. The layout display shows the angular ranges for each detector.

     

• Configure beam settings

  1. In TEMUI, go to the Tune tab and locate the Beam Settings panel.

  2. Select Probe mode.

     • Button is highlighted yellow when active.

  3. Select NanoProbe mode.

  4. Set spot number to 6.

     NOTE: NanoProbe provides a smaller, more coherent probe than MicroProbe. Lower spot numbers produce smaller probes with lower current; higher numbers produce larger probes with more current.

     TODO: Verify spot number convention for Spectra 300. On some Thermo Fisher systems, it is the opposite (spot 1 = most current).

     

1.4 Direct alignments

The basic alignments center the electron beam and align it through the optical column. Proper alignment is essential for optimal resolution and probe symmetry.

• Set magnification and open Direct Alignments

  1. Set magnification to 200-300kX using the magnification knob.

  2. In TEMUI, navigate to Tune tab, then Direct Alignments. This panel provides access to all fundamental beam alignment procedures.

  3. Select Diffraction Shift and Focus alignment to begin.

     

• Center ronchigram

  1. Observe the ronchigram position on the display. If the ronchigram is shifted from center, use the mulXY knobs to bring it back.

     

  2. The mulXY knobs now control diffraction shift. Adjust until the ronchigram is centered.

     

• Reset STEM AutoTuning

  1. In the quick dropdown menu, select STEM AutoTuning. This panel stores automatic alignment adjustments from previous sessions.

  2. Click Reset under Settings to clear stored values. This establishes a known baseline. Previous user adjustments persist and interfere with fresh alignments if not reset.

     

• Switch to probe image mode

  1. Press the Diffraction button on the hand panel to enter probe image mode (STEM scanning). The red light should turn off once pressed.

     No beam found? In the following step you will click Beam Shift and adjust the mulXY knobs. Watch the screen current: it changes from 0.000 nA to 0.001 nA, etc. This means you are shifting the beam position near the screen. You will see dim light coming from the edges. Press the Fine and Coarse buttons to adjust the sensitivity of the mulXY knobs.

     

     Diffraction mode vs. Probe image mode

     Mode	Probe	Display
     Diffraction mode	Stationary	Ronchigram - diffraction pattern from convergent probe
     Probe image mode	Scanning	STEM image - probe scans to build up image pixel by pixel

     The Diffraction button on the hand panel toggles between these two modes.

• Align beam shift

  1. Click on Beam shift in the Direct Alignments panel. The mulXY knobs now control alignment beam shift.

  2. Use the mulXY knobs to center the beam on the screen. The beam responds smoothly to knob movements. If the beam moves too quickly, press Fine on the hand panel to reduce sensitivity.

  3. Important: If the beam is lost after clicking beam shift, reduce magnification until the beam is visible, center using the mulXY knobs.

  4. Click Done once the beam is properly centered.

     

• Center C2 aperture

  1. Select Center C2 aperture from the alignment options. The system oscillates the C2 lens, causing the beam to expand and contract rhythmically.

  2. Watch the beam movement carefully. The beam pulses in and out. The goal is to make this expansion/contraction perfectly concentric (no lateral movement).

  3. Use the mulXY knobs to adjust the aperture position.

  4. Click Done when the movement is concentric.

     

• Align beam tilt

  1. Select Beam Tilt from the alignment options. This alignment minimizes the lateral shift of the beam when tilting.

  2. Use the mulXY knobs to minimize lateral movement of the beam. When properly aligned, the beam changes angle without shifting position.

  3. Reduce the lateral x and y movements as much as possible using the mulXY knobs.

  4. Click Done once the lateral movement is minimized.

     

• Verify final diffraction shift

  1. Press the Diffraction button on the hand panel to switch back to diffraction mode (view the ronchigram). The red light should now be on.

  2. Return to Diffraction Shift and Focus alignment for a final centering check.

  3. Use mulXY to center the ronchigram precisely on the display. Centering confirms the beam is on the optical axis.

  4. Click Done to complete the direct alignments.

     

Note: Mode changes (diffraction ↔ probe image, TEM ↔ STEM) disable descan. Re-enable descan after each mode switch. If the image looks distorted, verify Descan is enabled in STEM Imaging (Expert).

1.5 Monochromator tune

Before proceeding to probe correction, check that the monochromator is properly aligned and not partially blocking the beam. The monochromator selects a narrow energy spread from the electron source, improving resolution but reducing beam current.

• Open Monochromator Tune

  1. In TEMUI, go to the Mono tab and locate the Monochromator Tune (Expert) panel. This panel provides controls for adjusting the monochromator position and focus.

  2. Click on both Shift and Focus buttons to enable adjustment mode. At this point, the intensity knob controls monochromator focus and the mulXY knobs control monochromator shift.

     

• Adjust focus

  1. Adjust the intensity knob to bring the Focus value close to 0. As the monofocus approaches zero, the screen current increases because more electrons pass through the monochromator slit.

  2. While adjusting Focus toward 0, also adjust the mulXY knobs to ensure the beam isn’t blocked. The screen current in TEMUI should be above 15 nA when focus is near zero.

     No beam visible? Click Linear in the detector settings to switch from log to linear display mode. If the beam is still missing, return to the initial focus value and slowly bring it back toward 0 while adjusting mulXY, as in the Beam Shift alignment.

  3. Watch the current readout while adjusting.

     

• Center and adjust current

  1. Adjust the mulXY knobs to center the beam through the monochromator.

     

  2. Use the intensity knob to achieve the target beam current (~0.150 nA for high-resolution STEM).

     

• Deselect Shift and Focus

  1. Click Shift again to deselect both buttons (they toggle together).
  2. This returns the intensity knob and mulXY knobs to their normal functions. Verify the current readout shows the target value before proceeding.

• Re-verify eucentric height

  1. Use the z-axis controls to return to the “blow-up” point (eucentric height). Monochromator adjustments affect focus; re-verify eucentric height.

1.6 HAADF imaging setup

Before running aberration correction, set up HAADF (High-Angle Annular Dark Field) imaging to view the sample and find a suitable region. HAADF provides Z-contrast imaging where heavier atoms appear brighter.

• Switch to HAADF

  1. In the Velox acquisition software, click STEM to enter STEM mode,

  2. Click HAADF. This automatically inserts the HAADF detector.

  3. Verify in TEMUI that the HAADF detector shows “Inserted” status with the correct collection angle (63-200 mrad).

     

• Verify Descan is enabled

  1. In TEMUI, go to STEM Imaging (Expert) and verify Descan is enabled. Mode changes disable descan; re-enable after switching modes.

• Start live scanning

  1. Click the play button in Velox to start live scanning.

  2. The image is saturated (all white) initially. Detector signal adjustment follows in the next step.

     

• Adjust detector signal

  1. In Velox, click Scope tool to enable signal adjustment.

  2. Adjust Gain and Offset so the signal does not go above the dotted red lines.

     Signal math: Display = (Gain × Signal) + Offset

     • Gain (= Contrast): Multiplier that stretches the signal. 100% = no change, 200% = double the contrast
     • Offset (= Bias/Brightness): Shifts the baseline as a percentage of the display range

     

  3. If the signal is clipping at zero (bottom of display), increase Offset to shift the signal up.

     

  4. Adjust the magnification to ~20,000x using the magnification knob. Once adjusted, uncheck Scope tool to turn it off.

     

• Find sample boundary

  1. Reduce magnification to ~10,000x and navigate with the joystick to find a suitable region.

  2. Locate a boundary region with particles at the edge of a support film, with vacuum visible.

  3. This type of region provides excellent contrast for aberration correction.

     

• Adjust focus

  1. Once a suitable boundary is found, increase magnification to ~160,000x. Alternate between magnification and z-axis adjustments until focus is sharp.

     

  2. Adjust the z-axis while watching the HAADF image. Features become sharper as focus is approached.

     

  3. Continue adjusting magnification and z-axis.

     

  4. Finalize the position with sharp features and distributed particle sizes.

     

  Distributed particles are important for aberration measurement. Aberrations vary with position relative to the optical axis (e.g., coma increases further from center). The correction algorithm requires ronchigram data from multiple positions to accurately fit the aberration coefficients.

Part 2: Probe Correction

Before starting probe correction, retract the HAADF detector. C1A1 and Tableau both analyze the ronchigram, and the HAADF ring blocks high-angle electrons from reaching the camera below. In TEMUI, verify the HAADF detector shows “Retracted” status.

TODO: Confirm with staff whether HAADF must be retracted for C1A1/Tableau on Spectra 300 S-CORR.

Aberrations distort the electron probe and degrade image resolution. The goal of probe correction is to achieve a flat, aberration-free ronchigram. The figure below shows how individual aberrations affect the ronchigram appearance:

Interactive demo: Explore how aberrations affect the ronchigram at bobleesj.github.io/electron-microscopy-website/ronchigram

Probe correction uses two tools in the Probe Corrector S-CORR software:

The correction workflow:

The following workflow is covered in this section. Follow the steps below, then use this diagram as a quick reference:

2.1 C1A1 correction

C1A1 corrects first-order aberrations: defocus (C1) and 2-fold astigmatism (A1). These are the dominant aberrations that must be corrected before higher-order Tableau measurement. The C1A1 procedure analyzes the ronchigram to measure and correct these aberrations iteratively.

• Open Probe Corrector

  1. On the top left monitor, open the Probe Corrector S-CORR software (main interface for aberration measurement and correction).

  2. Check the mode indicator in the top right. Verify it shows STEM@300KV:

     

  3. If MC_STEM@300KV appears instead, the system is in monochromated STEM mode. Follow the steps below to reset to standard STEM mode. If STEM@300KV is displayed, skip ahead to “Configure C1A1 options.”

     

  4. To reset, in TEMUI go to Mono, then open Monochromator (Expert) and click Filter:

     

  5. Then click Unfilter to reset to standard STEM mode:

     

• Configure C1A1 options

  1. In the Probe Corrector software, click Options to expand the configuration panel:

     

  2. Set Probe semi aperture to 30 mrad:

     

• Switch to diffraction mode

  1. Stop live scanning in Velox by clicking the play button, then ensure Diffraction mode is on on the hand panel. C1A1 analyzes the ronchigram, so diffraction mode (stationary probe) is required, not probe image mode (scanning):

     

  2. Click the Beam Blank button to unblank the beam. Stopping the scan automatically blanks the beam. The Probe Corrector software requires an unblanked beam to read the ronchigram:

     

• Run C1A1

  1. Go to the C1A1 tab in the Probe Corrector software. Before clicking Start, verify the ronchigram is visible on the left monitor:

     

  2. Click Start to begin aberration measurement. The software continuously analyzes the ronchigram and displays measured aberration values (C1, A1, A2, B2, WD) in the table.

  3. Set Auto correct to 100% for the first iteration.

  4. Click 0th-2nd to apply corrections for all first and second order aberrations.

     

• Iterate C1A1

  1. Click 0th-2nd repeatedly to apply corrections. Each row represents one measurement cycle. Watch the aberration values decrease with each iteration.

     

  2. Reduce the Auto correct percentage to 75% after several iterations (typically 3 to 5) to prevent overcorrection.

     If A1 is still high but other values are good, click A1 specifically to correct only astigmatism.

     

  3. When to stop: C1A1 values are stable when they no longer decrease significantly between iterations. Target: C1 (defocus) < 1 nm and A1 (astigmatism) < 3 nm. Click Stop when values are stable.

2.2 Tableau measurement

Tableau measures higher-order aberrations (A2, B2, C3, S3, A3) by acquiring ronchigram patterns at multiple beam tilt angles. The software analyzes how the ronchigram changes with tilt to extract the full aberration function. Tableau is more comprehensive than C1A1 and necessary for highest resolution.

• Open Tableau tab

  1. Switch to the Tableau tab in the Probe Corrector software for full aberration measurement and correction.

  2. Select Standard for Tableau type. This acquires a sufficient number of tilt positions for accurate measurement without taking excessive time.

  3. Set the Outer tableau tilt to 40 mrad. Larger tilts probe higher-order aberrations but require more time.

  4. Verify the Probe semi aperture is set to 30 mrad to match the beam settings.

     

  5. Click Options and select the A5 toggle. It measures up to 5th order aberrations.

• Run Tableau measurement

  1. Click Start to begin the Tableau measurement. The software automatically tilts the beam to multiple angles and acquires ronchigram images at each position.

  2. Wait for measurement completion. The ronchigram shifts across the screen as the software captures patterns at different tilts and focus levels (under-focus and over-focus at each tilt). This movement is expected.

  3. Do not modify the stage position during measurement. If the beam is unstable, stop and ask staff.

     

• Accept measurement

  1. Click Accept after measurement completes. This validates the data for corrections.

• Review measurement results

  1. Click the State of correction tab. This shows all measured aberration coefficients in three columns:

     • Estimation: Just measured values
     • Latest accepted measurements: Previously applied corrections (yellow = outside limits)
     • Estimation in image coordinate system: Values transformed to image coordinates
     

  2. Check the phase plate visualization on the right. A well corrected probe has a flat, symmetric phase plate. Strong asymmetric patterns indicate uncorrected aberrations:

     

• Apply corrections

  1. Set Auto correct to 75% to prevent overcorrection. Yellow highlighted values in the “Latest accepted measurements” column indicate aberrations outside acceptable limits. Correct these first. In this example, S3 (1.167 μm) and C3 (-2.553 μm) are highlighted yellow:

     

  Note: either clicking B4 or D4 can have a significant impact on C1 and A1 values.

  2. Click the aberration buttons at the bottom to apply corrections. The phase plate visualization shows the limiting aberration. Correct this one first. Click the button repeatedly until the value improves sufficiently, then move to the next limiting aberration.

  3. The “Changes” column tracks how many corrections have been applied. After correcting S3 and C3, the values improve significantly:

     • S3: 1.167 μm → 72.93 nm
     • C3: -2.553 μm → -159.6 nm
     

• Run full measurement

  1. After applying corrections, run another complete Tableau measurement to verify the improvements.

  2. Check the aberration surface and phase plate displays. A well-corrected probe shows:

     • Flat aberration surface with green in the center (minimal phase variation across the aperture)
     • Symmetric phase plates without strong directional features
     

  Target values (30 mrad semi-aperture):

  Parameter	Target
  C1	< 1 nm
  A1	< 3 nm
  A2	< 40 nm
  B2	< 25 nm
  C3	< 1.5 μm
  A3	< 1 μm
  S3	< 500 nm

  TODO: CONFIRM WITH ANDREW for IDEAL TOTAL D50

• Verify with C1A1

  1. Return to the C1A1 tab in the Probe Corrector software. Tableau correction can sometimes introduce small first-order errors.
  2. Click Start to begin C1A1 measurement again.
  3. Click A1 to correct any residual astigmatism introduced by Tableau.
  4. Click 0th-2nd if defocus also needs adjustment.
  5. Iterate between Tableau and C1A1 if necessary until all values are within specification.

• Check resolution

  1. The State of correction panel displays resolution estimates on the right side: Total D50 and Optimum D50. D50 represents the probe diameter containing 50% of the beam intensity (smaller = better resolution).

     

     TODO: CONFIRM WITH ANDREW

  2. Target: Total D50 of 70-75 pm for high-resolution STEM imaging. The Optimum D50 shows the theoretical best achievable with current aberrations. If these values match closely, corrections are complete.

     

  3. If D50 values are significantly higher than target, continue iterating: run another Tableau measurement, apply corrections, then verify with C1A1. The image below shows C1A1 iterations after Tableau corrections:

     

• Return to probe image mode

  1. Once correction is complete, press the Diffraction button on the hand panel to switch back to probe image mode (STEM scanning).
  2. The system is now ready for high-resolution image acquisition.

Part 3: Imaging

3.1 Acquire images

With aberration correction complete, the system is ready for high-resolution STEM image acquisition. The probe is optimized for atomic-resolution imaging.

• Acquire HAADF image

  1. In Velox, click STEM to enter STEM mode, then click HAADF to select the HAADF detector.

  2. Click the play button to start live scanning. Image quality is noticeably improved compared to before correction. A well-corrected probe produces sharper, more detailed images.

  3. For initial survey imaging, set resolution to 1024×1024 and dwell time to 500 ns. Fast scanning enables navigation while maintaining image quality.

     

• Navigate to area of interest

  1. Use the live scan to find the region of interest. Use the joystick or click on the image to move to different regions. With a well-corrected probe, atomic lattice fringes are visible in crystalline materials.

     

  2. Adjust focus using the z-height controls if needed. Small focus changes can significantly affect atomic-resolution contrast.

     

• Capture high-resolution scan

  1. Increase the resolution to 2048×2048 or higher. Check the Velox toolbar to verify resolution and dwell time settings before starting the acquisition.

     

  2. Increase the dwell time to 5 µs for better signal-to-noise ratio. Longer dwell times collect more electrons per pixel, reducing noise but increasing total scan time and potential for drift artifacts. After acquisition completes, the beam is blanked automatically to prevent sample damage.

     

3.2 Fine-tuning with Sherpa

Sherpa provides rapid aberration correction that is faster than full Tableau measurement. Use Sherpa for quick refinements after the main alignment, or when aberrations drift during extended imaging sessions.

• Prepare for Sherpa

  1. Before running Sherpa, verify the ronchigram is centered. Press the Diffraction button on the hand panel to switch to diffraction mode (view the ronchigram).

  2. In TEMUI, go to Direct Alignments and select Diffraction Shift and Focus alignment:

     

  3. Use the mulXY knobs to center the ronchigram on the display. A centered ronchigram ensures Sherpa measurements are accurate.

     

• Adjust C2 aperture (optional)

  1. To change C2 aperture size (for example, switching to 50 µm for different probe conditions), locate the Apertures panel and change Condenser 2 from 70 to 50 (or the desired size).

     

  2. Click Adjust to center the new aperture. The beam remains centered when changing aperture sizes. If not centered, use the adjustment controls to re-center.

     

• Open Sherpa

  1. Open the Sherpa software. Sherpa displays the HAADF image with a crosshair marker indicating the measurement region.

     

  2. Click C1/A1 to run first-order correction (defocus and 2-fold astigmatism).

     

• Run B2/A2 tuning

  1. After C1/A1 completes, click the B2/A2 button to correct second-order aberrations (axial coma B2 and 3-fold astigmatism A2).

  2. Wait for the tuning to complete. Sherpa acquires images and determines optimal corrections.

  3. Run multiple iterations if the first pass does not achieve optimal results.

     

• Review results

  1. Sherpa displays the initial image alongside the optimized image for comparison. The corrected image shows improved sharpness and resolution.

     

3.3 Load your own sample

After completing probe correction on the standard sample, follow the below steps unload the current sample standard and load your own. For holder-specific instructions (single-tilt, double-tilt, tomography), see Sample Loading.

• Remove the standard sample

  1. Put on gloves before handling any holders or samples.

  2. Blank the beam and verify the screen is inserted. The screen protects the detectors and cameras below from the beam.

  3. Close the column valves by pressing Column Valves Closed.

     If a “VCP” error occurs, follow the instructions on the Spectra NEMO page.

  4. Reset the holder by clicking reset on the Stage menu.

     

  5. Confirm the stage x, y, z values are returning to zero after you reset the holder stage.

  6. Pull the holder straight out to the first resistance point. Do not force beyond this point. Turn clockwise, then pull the rest of the holder out continuously.

• Load your sample and insert the holder

IMPORTANT: Do not remove the standard sample from the single-tilt holder. Use a separate holder to load your sample.

1. For holder-specific loading instructions, see Sample Loading.

2. Align the holder with the blue line on the goniometer.

   

3. Push the holder in until you feel resistance. Do not push all the way in.

4. The turbo pump starts automatically. Wait ~2 minutes for pressure to stabilize. You can monitor the time in TEMUI or on the screen attached to the Spectra instrument.

   

   Why wait? The holder insertion opens a small chamber to atmosphere. The turbo pump must evacuate this air before you can insert the holder into the main column. Rushing this step would introduce air into the ultra-high vacuum column, potentially damaging the electron gun and contaminating the system.

5. Turn the holder counter-clockwise until you feel gently stuck, then guide the holder to push in. The holder should move in smoothly.

   

6. In TEMUI, turn off the turbo pump. Confirm the holder type when prompted.

• Re-do eucentric height

  1. Open column valves and re-do eucentric height for your new sample (1.2). Each sample sits at a different physical height in the holder. Find the ronchigram “blow-up” point again so the sample stays centered when tilted and the probe is properly focused.
  2. Run a quick C1A1 or Sherpa to verify probe correction still holds after the sample change.

3.4 End session

• Reload the standard sample

  1. Put on gloves before handling any holders or samples.

  2. Blank the beam and verify the screen is inserted.

  3. In TEMUI, click Column Valves Closed.

  4. Click Reset Holder under the Stage menu. Visually verify that the X, Y, and Z stage coordinates are reset after the button is pressed.

     

  5. Pull the holder with your sample straight out to the first resistance point. Do not force beyond this point. Turn clockwise, then pull the rest of the holder out continuously.

  6. Set aside your holder and pick up the single-tilt holder with the standard sample.

  7. Push the single-tilt holder with the standard sample in until you feel resistance. Do not push all the way in.

  8. The turbo pump starts automatically. Wait ~2 minutes for pressure to stabilize.

  9. Turn the holder counter-clockwise until you feel gently stuck, then guide the holder to push in.

  10. In TEMUI, turn off the turbo pump. Confirm Single tilt on TEMUI.

Checklist before leaving the Spectra room

• Beam is blanked.
• Reset Holder has been pressed and X, Y, Z stage coordinates are verified reset.
• Stage is returned to 0° tilt (alpha and beta).
• Arina detector is retracted, verified on the left hand panel.
• Arina detector is turned off, verified on the local Firefox URL.
• INT SCAN physical button is in pressed state.
• Screen is inserted.
• Column valve is closed.
• Turbo pump is turned off.
• Standard sample is loaded.
• All holders are capped and stored in the holder box.
• The sample loading area is tidy.
• Spectra usage is terminated on NEMO.
• Internet accounts (Google, Outlook, etc.) are signed off.
• Fill out the logbook if anything unusual happened during the session.

Troubleshooting

Common problems encountered during STEM sessions.

FAQ

Beam blanking

When the beam is blanked, the electron beam is deflected away from the sample so no electrons hit it. This prevents unnecessary radiation damage to the sample when not actively imaging. The beam is automatically blanked when scanning stops or after taking a picture. Manual blank/unblank is available via the Beam Blank button on the hand panel or in the software.

Monochromator focus adjustment

The monochromator filters the energy spread of the electron beam by passing it through a narrow slit. Setting Focus = 0 places the beam crossover exactly at the monochromator slit plane. This position maximizes electron throughput while maintaining energy filtering. If the focus is offset from zero, the beam crossover occurs before or after the slit, reducing beam current and degrading energy resolution.

Appendix

Aberration notation

Different notations exist for aberrations in the literature. The table below shows the Krivanek notation (used in Probe Corrector software), the alternative notation (used in this guide), and descriptions.

Changelog

• Mar 1, 2026 - Add pre-session checklist, sample loading section with glove requirement, end session with explicit reload steps, fix image paths
• Feb 28, 2026 - Add prerequisite link to TEM Alignment guide; add lost-beam troubleshooting
• Jan 31, 2026 - Initial draft: instructions by Sangjoon Bob Lee, screenshots by Andrew Barnum during Spectra 300 hands-on training