Electron Microscopy (WIP)

Step-by-step visual guides for learning TEM. Focused on practice sitting in front of the microscope with visual elements.

WIP:

• Use real screenshots instead of pictures
• Better pictures: proportion, focus, FOV
• More visuals added to each session when needed

Disclaimer: Always follow https://barnum.su.domains/ for correctness. Only use this documentation if you are working with the authors and need quick visual references.

Available guides

Instrument links:

• FEI Titan at SNSF
• Spectra 300 at SNSF

Other resources:

• Ronchigram simulator
• (S)TEM alignment diagrams

Looking for volunteers!

We appreciate feedback, corrections, and contributions from the community!

• Found an error? Open an issue or submit a PR
• Want to add your institution’s SOP? Reach out to @bobleesj — we can help with writing and formatting as long as you have notes, Word docs, or rough drafts
• Have suggestions? See the GitHub repo for contribution guidelines

Acknowledgments

Authors thank Dr. Pinaki Mukherjee and Andrew Barnum for training @bobleesj and Guoliang Hu at Stanford SNSF.

Changelog

• Mar 1, 2026 - Restructure STEM and TEM guides, add pre-session checklist and end session procedures
• Feb 28, 2026 - Reorganize guide structure, rename directories, add safety warnings
• Jan 31, 2026 - Add STEM probe correction guide with screenshots from Andrew Barnum training
• Jan 31, 2026 - Add draft Titan TEM guide
• Dec 18, 2025 - Add EELS and EDS guides
• Dec 17, 2025 - Use mdBook to render static pages and host on GitHub
• Dec 14, 2025 - Begin Electron Microscopy training documentation, led by @bobleesj

Sample loading

Location

TITAN:

Sample loading is done outside of the room where Titan is hosted:

Spectra 300:

Sample loading can be done inside the Spectra room:

Sample holders are stored here at room temperature:

Single-tilt holder

Load sample:

FIXME: use technical terms for these objects… “pin” or “clip”?

1. Push the pin inside the tiny hole shown below:

   

2. Lift the clip gently:

   

3. If using a copper grid, pinch the tip of the copper grid

   

4. Place the sample, shiny side up for the standard sample

   

Double-tilt holder

Load sample:

1. Load the sample and washer (gold donut):

   

2. Add the cap and rotate the holder about the long axis to ensure the sample is secure:

   

Unload sample:

1. Press down the very small hole gently as shown below:

   

2. The three parts should all be disassembled and placed on the bottom:

   

Tomography holder

Unload sample

1. The sample grid is held between the two “arms” of the metal strips.

   

2. Rotate the screw counterclockwise about 90 degrees. Repeat for the other “arm”.

   

3. Move the two strips away from each other to free the sample

   From:

   

   To:

   

4. Remove the sample

   

Load sample

Follow the process in reverse: load a new sample, pull the two strips closer together, and rotate the screws clockwise.

Changelog

• Dec 20, 2025 - Rnhance holder insersion process with visuals
• Dec 18, 2025 - Reorganize into Start/End session sections for use across all tutorials by @bobleesj
• Dec 17, 2025 - Add sample loading region by @bobleesj
• Dec 15, 2025 - Add tomography holder section by @bobleesj, with images taken by Guoliang Hu
• Dec 13, 2025 - Add single-tilt, double-tilt holder section by @bobleesj, with images taken by Guoliang Hu

TEM (Titan)

Coming soon.

This guide covers TEM alignment and imaging on the FEI Titan at Stanford SNSF.

Changelog

• Feb 28, 2026 - Add Titan instrument photos

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

TEM (Spectra)

This guide covers optional TEM alignment on the Spectra 300: column setup, eucentric height, aperture alignment, and image correction. TEM mode is useful for fast sample navigation and for users who need TEM-specific data (HRTEM, diffraction patterns). Most users will proceed directly to STEM (Spectra).

Prerequisite: The sample is already loaded and the holder is inserted into the Spectra 300. For sample loading and end session procedures, see STEM (Spectra).

Acronyms:

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

Workstation layout:

Overview

This guide covers two main phases:

Part 0: Safety check

Complete the pre-session checklist in STEM (Spectra) before proceeding. Do not skip this step.

Part 1: Column alignment

1.1 Open column valves

Before imaging, verify that the vacuum system is ready and the column valves can be safely opened.

• Verify vacuum pressure

  1. In TEMUI, check the vacuum pressure values on the log scale (lower = better):

     Gauge	Log Value	Why Important
     Gun	1	Highest vacuum needed for stable electron emission
     Liner	<10	Prevents electron scattering along beam path
     Octagon	1	Protects sample from contamination and oxidation
     Projection	<30	Maintains image quality in projection system
     Buffer tank	<50	Ensures stable pumping performance
     Backing line	<80	Turbo pump pushes compressed gas into the backing line

• Open column valves

  1. In TEMUI, click Col Valves Open. The status changes to indicate the column valves are open and the turbo pump is off.

     

• Set condenser apertures

  1. In TEMUI, go to the Tune tab, then Apertures. Set Condenser 1, 2, 3 to 2000, 70, 1000.

     

1.2 Beam setup

Configure the beam parameters for initial navigation and sample finding.

• Enter TEM mode

  1. On the Velox software (bottom right monitor), verify TEM mode is active. If not, click the TEM button.

• Set spot size

  1. Set Spot Size 3 by pressing the L3 or R3 button on the hand panel. As spot size decreases, screen current increases and the image gets brighter.
  2. If the image is too bright, turn the intensity knob to decrease the screen current to around 2 nA and press Linear mode to see better contrast.

• Find sample region

  1. Set ~500x magnification by adjusting the magnification knob.

  2. Locate the gold (dark) and amorphous carbon boundary by driving the joystick on the hand panel. This contrast boundary serves as a visual marker to identify the region of interest across various magnifications.

     

1.3 Eucentric height

At eucentric height, the sample remains stationary when tilted. This is essential for accurate imaging and aberration correction. Complete eucentric height alignment after loading each sample. Do not skip this step.

• Adjust z-axis

  1. Set ~7,500x magnification by adjusting the magnification knob.

     

  2. Press z-axis up or down on the hand panel. Watch the image contrast change as the sample moves through focus. At eucentric height, the contrast is minimized (the image appears most “washed out”).

     

1.4 Monochromator tune

The monochromator selects a narrow energy range from the electron beam. If the beam edge looks jagged, the monochromator needs alignment.

• Check beam edge

  1. Do you see a jagged area along the beam edge in the previous step? If not, skip this section. Otherwise, follow the steps below.

• Adjust monochromator

  1. In TEMUI, go to the Mono tab, then open Monochromator Tune (Expert) and click Shift.

     

  2. Adjust mulXY knobs until the jagged area disappears.

1.5 C2 aperture alignment

The C2 aperture controls convergence angle and beam size. It blocks off-axis electrons — only electrons within a certain angular range pass through. A user must center this aperture on the optical axis so that the beam expands and contracts symmetrically.

• Enter two-lens mode

  1. In TEMUI, go to the Tune tab, then Beam Settings, and click Twolens. In two-lens mode, C3 is turned off, so the beam behavior on screen is purely from C2. This makes it straightforward to detect and correct any C2 aperture misalignment. In three-lens mode, C3 reshapes the beam after C2, masking the misalignment.

     

• Center and align C2 aperture

  1. Center the beam by rolling the hand panel ball.

  2. Converge the beam by varying the intensity knob.

  3. Vary beam size by turning the intensity knob counterclockwise and clockwise. Notice the beam expansion is not concentric — this indicates the C2 aperture is off-center.

  4. Make the beam concentric: go to Apertures, click Adjust next to Condenser 2, then adjust the mulXY knobs until the beam expands and contracts concentrically.

     

• Return to three-lens mode

  1. In TEMUI, go to Beam Settings and click TEM to return to three-lens mode.

     

  2. Verify the beam is centered and concentric.

     

1.6 Condenser stigmatism

Condenser astigmatism causes the beam to appear elliptical instead of round. Correcting this ensures a symmetric probe.

• Increase magnification

  1. Set ~200kx magnification by adjusting the magnification knob.
  2. If the beam has shifted from center, go to Tune tab, then Direct Alignment, click Beam Shift, and adjust the mulXY knobs to re-center.

• Correct stigmatism

  1. Enlarge the beam by adjusting the intensity knob.

     

  2. In TEMUI, click Stigmator, then Condenser. Adjust the mulXY knobs to make the beam as round as possible. The beam should remain circular as you vary the intensity knob. Press None when done.

1.7 Beam tilt

Beam tilt alignment minimizes lateral beam shift when the beam angle changes. Proper alignment ensures the beam tilts around a single point without drifting.

• Align beam tilt

  1. In TEMUI, go to Direct Alignment and click Beam tilt pp X. Adjust the mulXY knobs to minimize the lateral jiggle.
  2. Repeat for Beam tilt pp Y.
  3. If the beam center has shifted, click Beam Shift and adjust the mulXY knobs to re-center.

1.8 Rotation center

Rotation center alignment ensures the image rotates around the center of the field of view when focus changes.

• Align rotation center

  1. In TEMUI, go to Direct Alignment and click Rotation Center. The image pulses in and out of focus.
  2. Adjust the mulXY knobs to minimize lateral movement. The pulsing should appear concentric (expanding and contracting from the same point) with no side-to-side drift.

Part 2: Image correction

2.1 Capture image

Before running the image corrector, a user must set up live imaging in Velox and find a suitable sample region.

• Prepare for imaging

  1. Find a flat area with a distribution of particle sizes and no holes.
  2. Important: Enlarge the beam to cover the entire fluorescent screen before lifting it. When the screen is raised, the camera and detectors below are exposed to the beam. A concentrated beam can permanently damage them.
  3. Press R1 on the hand panel to lift the fluorescent screen.

• Start live imaging

  1. In Velox (right monitor), click the play button to start live imaging.

     

  2. Do not change the intensity knob while the screen is lifted. The screen is lifted when TEMUI shows a black display with dose reading “Unavail”.

  3. Gold nanoparticles should be visible on screen.

     

• Explore focus (optional)

  1. Press the z-axis buttons to observe how focus affects the image.

     Underfocus — edges appear bright with white Fresnel fringes:

     

     On focus — minimal fringe contrast:

     

     Overfocus — contrast inverts, dark fringes at edges:

     

2.2 C1A1 correction

C1A1 corrects first-order aberrations in the image-forming lenses: defocus (C1) and 2-fold astigmatism (A1).

• Set underfocus

  1. Press Z-axis down until you see 4-5 rings in the FFT (slight underfocus). The rings indicate Thon rings from the amorphous carbon, which the corrector software uses for aberration measurement.

     

• Reset stigmator values

  1. Stop live scanning by clicking the play button in Velox.

  2. In TEMUI, go to the Stigmator quick tab. Reset Objective and Image A1 to zero. If non-zero, right-click each button to reset, then click Done.

     

• Run C1A1

  1. Open the ImageCorrector software (top left monitor).

  2. Set exposure time to 0.3s.

  3. Go to the C1A1 tab and click Start. The microscope wobbles the focus up and down (changing objective lens current). The FFT is captured and its ring symmetry, angular distribution, and ring spacing are analyzed.

  4. During the iteration, carefully set intensity to 800–900 counts by adjusting the intensity knob so the corrector has enough signal.

     

  5. Under Auto correct, set to 75%, then press Focus and A1 during the iteration to apply corrections.

  6. Aim for A1 < 5 nm. If C1 shows orange, manually adjust the Z-axis during the iteration. C1 should be close to the suggested value (in the image above, the software suggests C1 of −599.3 nm).

2.3 Tableau measurement

Tableau measures higher-order aberrations by acquiring images at multiple beam tilts. This is necessary for sub-angstrom resolution.

• Run Tableau

  1. In ImageCorrector, go to the Tableau tab, select Standard next to Tableau type, then click Start.

     

• Verify results

  1. After the iteration completes, verify the aberration values match the targets below, then click Accept:

     Parameter	Resolution < 0.10 nm (20 mrad)	Resolution < 0.08 nm (24 mrad)
     A1	< 5 nm	< 5 nm
     A2	< 100 nm	< 50 nm
     B2	< 100 nm	< 50 nm
     C3	~ −8 μm	~ −8 μm
     A3	< 5 μm	< 1.5 μm
     S3	< 5 μm	< 1 μm

  2. In Velox, click the camera button to capture an image and verify improvements.

2.4 Save optics settings

• Save register

  1. In TEMUI, go to Files, then SBL FEG Registers.

  2. Add name 300KV-TEM-<NAME> and click Add.

     

• Verify corrected image

  1. In Velox, click the Play button to start live imaging and verify the aberration-corrected image quality.
  2. Done. You are now ready for STEM probe alignment.

Appendix

Save file to USB

Plug your USB into the following computer:

Reference images (click to expand)

Gray colors during C1A1 probe correction:

Seeing gray colors like below?

In Velox, click Auto-tune. Increase the signal until it touches the red and blue dotted lines:

Hand panel R1, R2, R3 values:

Stage position and coordinates:

Dose rate and TEM mode display:

HAADF detector on TEMUI:

Samples with holes:

Wobbler to check eucentric height:

At eucentric height, tilting the holder should induce minimal shift.

Smart tilt:

Beam setting in Quick tab:

Stage piezo in Quick tab:

Stage tab:

Troubleshooting

Common problems encountered during TEM sessions.

FAQ

Convergence angle: In TEMUI, go to Beam Setting, then Probe, and use the mulXY knobs to adjust.

Tableau and C1A1: Tableau measures aberrations visually across multiple tilt angles. C1A1 corrects first-order aberrations (defocus and astigmatism). Run C1A1 first, then Tableau for higher-order corrections.

Underfocus direction: Counterclockwise on hand panel, Z-axis down.

Eucentric height: The z-position where tilting does not shift the sample. At eucentric height, defocus = 0 and probe size is smallest relative to the sample.

Beam Shift vs hand panel ball: Beam Shift stores the center position internally, so the beam stays centered when changing magnification. The hand panel ball moves the beam but does not save the position.

Underfocus vs overfocus: Underfocus produces bright white Fresnel fringes at edges. Overfocus inverts the contrast with dark fringes.

Monochromator: Filters the electron beam to select a narrow energy range, improving energy resolution for EELS and reducing chromatic aberration.

Two-lens vs three-lens mode: Two-lens mode (C1+C2) turns off C3, providing a simpler beam path for C2 aperture alignment. Three-lens mode (C1+C2+C3) is the standard operating mode for TEM imaging.

Objective lens in TEM: In TEM, the objective lens sits below the sample and forms the first magnified image. In STEM, it sits above the sample and focuses the probe.

C2 aperture purpose: The C2 aperture blocks off-axis electrons, controlling the convergence angle and beam current. It must be centered on the optical axis for symmetric beam expansion.

References

• Thermo Fisher Spectra 300 TEM

Changelog

• Mar 1, 2026 - Restructure to match STEM guide format with subsections, checklists, and troubleshooting table
• Dec 15, 2025 - Add pre-probe corrector with STEM Direct Alignment steps by @bobleesj
• Dec 12, 2025 - Add STEM training images by Guoliang Hu
• Dec 8, 2025 - First draft of Spectra training by @bobleesj

4DSTEM

This guide covers 4DSTEM data acquisition using the Dectris Arina detector on the Spectra 300 at Stanford SNSF. 4DSTEM records a full convergent beam electron diffraction (CBED) pattern at every scan position, producing a 4-dimensional dataset (2D scan x 2D diffraction). Screenshots recorded by Guoliang Hu during training; instructions written by Sangjoon Bob Lee.

Prerequisite: Complete TEM (Spectra) column alignment and STEM (Spectra) probe correction before starting this guide.

Acronyms:

• mulXY - Multifunction X/Y knobs on hand panel
• TEMUI - TEM User Interface (software)
• CBED - Convergent Beam Electron Diffraction

Overview

Part 1: Detector setup

1.1 Retract CETA detector

Before inserting the Arina detector, a user must retract the CETA camera. Both detectors occupy the same physical space below the column. If the CETA is not retracted, inserting the Arina will crash both detectors. Do not skip this step.

1. On the bottom left computer, open the blanker/shutter software (red square icon with white T).

2. Click the CETA icon to retract the CETA detector.

   

3. Visually verify the CETA camera position is retracted from the diagram.

4. In TEMUI, locate the STEM Detector (User) panel and verify all detectors are retracted:

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

1.2 Initialize the Arina detector

1. Open the instrument enclosure on the Spectra 300.

   

2. Locate the Dectris Arina detector unit inside the enclosure.

   

3. Press and hold the button below the Arina detector (blue indicator light) for 10 seconds. When powered on, the button stays pressed in and the blue light is illuminated.

1.3 Connect remote software

1. On the control workstation, open Firefox and click the remote connection bookmark.

   

2. Enter the detector IP address: 192.168.12.73.

3. Click Initialize detector. Wait for initialization to complete before proceeding. The interface shows a progress bar while the detector initializes.

   

1.4 Configure file saving

1. Open the NOVENA detector software.

2. Click Save Images and select the destination folder.

   

3. Set the filename format to (name)_%00%. The %00% placeholder auto-increments the frame number.

4. Use Continuous for live streaming (preview) and Single to record and save a dataset.

Part 2: Beam configuration (optional)

The STEM (Spectra) guide sets 30 mrad convergence angle by default. If that is suitable for your experiment (e.g., ptychography), skip this section and go directly to Part 3: Acquisition. If you need a different convergence angle (e.g., nanobeam diffraction), follow the steps below.

2.1 Enable descan

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

2.2 Configure beam for nanobeam diffraction

The default STEM setup uses C2 = 70 and 30 mrad convergence angle. For nanobeam diffraction, reduce both to get separated Bragg disks. The table below shows typical values:

1. In TEMUI, go to Tune tab, then Apertures. Change C2 from 70 to 50, and C3 from 1000 to 30.

   

2. In Beam Setting, click MF-Y Convergence Angle. Use the mulY knob to adjust the convergence angle to 10 mrad, then click MF-Y Convergence Angle again to deselect.

   

3. Adjust beam current: in TEMUI, go to Mono, click Focus, and use the intensity knob to set the current to ~0.032 nA.

4. Set the camera length to 230 mm (or 285 mm depending on the required angular range for your material).

2.5 Retract HAADF

1. In TEMUI, retract the HAADF detector. The HAADF ring would block electrons from reaching the Arina detector below.

Part 3: Acquisition

3.1 Insert detector and configure scan

1. On the Arina hand panel, press Insert to move the detector into position. The green “Inserted” light confirms the detector is in place.

2. On the scan control box, press EDS Scan.

   

3. Press R1 on the hand panel to lift the fluorescent screen. The Arina detector sits below the screen.

3.2 Acquire data

1. In the NOVENA software, click Scan, then Continuous to start a live preview. Verify the central beam is centered on the detector.

2. If the beam is off-center, use the mulXY knobs with diffraction shift to center it.

3. Once centered, click Stop, then click Single Scan to acquire and save the dataset.

   NOTE: Each scan produces a 4D dataset: a CBED pattern at every pixel in the scan area. File sizes can be large depending on scan resolution and detector binning.

3.3 Quick analysis

1. In the NOVENA software, use Rebin and Reprocess for a quick check of the acquired data. For detailed analysis, export the data for processing with external software (py4DSTEM, etc.).

Part 4: End session

4.1 Retract the Arina detector

1. On the Arina hand panel, press Retract to move the detector out of the beam path. The green “Retracted” light confirms the detector is clear.

   

4.2 Power off the detector

1. Open the Spectra 300 instrument enclosure.
2. Press and hold the button below the Arina detector for 10 seconds. The button releases and the blue indicator light turns off.

4.3 Close session

Follow the steps in End session.

Changelog

• Feb 28, 2026 - Rewrite SOP by Sangjoon Bob Lee with full procedural instructions, inline FAQ dropdowns, and new images
• Dec 10, 2025 - First draft and images shared by Guoliang Hu

EELS

Caution

VERY ROUGH DRAFT - @bobleesj and Guoliang Hu took notes and pictures during training. This document will be updated with more detailed steps and images.

TODO:

• Add image for “EELS Scan” button (Step 2, Part 3)
• Better photo for STEM SI button
• Show what auto gain looks like
• Clarify zero loss extraction steps
• Add comparison images for beam not centered

This guide covers Electron Energy Loss Spectroscopy (EELS) on the Spectra 300. The process has two parts: calibration in TEM mode and STEM EELS spectrum imaging.

Prerequisite: Complete the STEM alignment procedure before starting.

Acronyms:

• GIF - Gatan Imaging Filter
• EFTEM - Energy Filtered Transmission Electron Microscopy
• ZLP - Zero Loss Peak
• SI - Spectrum Imaging
• mulXY - Multifunction X/Y knobs on hand panel

Part 1: Calibration

Use a vacuum or thin amorphous carbon area for calibration.

1. Find a calibration area

   • Locate a vacuum region or thin amorphous carbon area on the standard sample

2. Open DigitalMicrograph

   • Open DigitalMicrograph on the left monitor

   • If you see any dialog box, click OK to dismiss

     

   • Click EFTEM (Energy Filtered Transmission Electron Microscopy)

3. Open FilterControl

   • Go to Help → User Mode → Power User

   • Go to Window → Floating Window → Filter Control

     

   • Notice the green circle in TEMUI showing EELS detector is active

     

4. Set beam intensity

   • Converge the beam by adjusting the intensity knob

     

   • Lift the fluorescent screen by pressing R1

   • Click View in DigitalMicrograph

   • Go to Filter Control → Aperture → Mask to verify beam position

     Correct intensity:

     

     Too high intensity (oversaturated):

     

5. Center and tune the GIF

   • Click Center ZLP in Filter Control

   • Click Tune GIF. Notice the message appears:

     

   • Click OK to confirm

Part 2: TEM EELS acquisition

1. View the sample

   • Set magnification to ~17,000x

   • Click View in DigitalMicrograph

   • Select EF-CCD Camera → View to see image real-time

     

2. Acquire zero loss image

   • Go to SingleMap → click Zero Loss Image

     

3. Switch to EELS mode

   • Click EELS button to switch modes

     

     FIXME: use image where EELS is clicked

   • Notice the 2D EELS spectrum. Observe the plasma peak near the zero loss peak.

     

   FIXME: what’s plasma peak?

4. Align the zero loss peak

   • Set exposure to 2e-4 in View mode

   • Click Align ZLP

     

     FIXME: where is ZLP click?

Part 3: STEM EELS spectrum imaging

1. Set camera length

   • In Velox, change camera length to 29 mm or 37 mm

   • Notice the beam size decreases

     

2. Enable EELS scanning

   • Press EELS Scan in the software

   FIXME: attach image

3. Find a vacuum area

   • Navigate to a vacuum region for initial alignment

4. Center the beam

   • In EFTEM mode, use mulXY knobs to center the beam

     Before centering:

     

     After centering:

     

5. Switch to STEM SI mode

   • Click STEM SI to switch to Spectrum Imaging mode

     

6. Find sample area

   • Navigate to an area of interest on your sample

7. Start scanning

   • Click Scan → View to see the image

     

8. Adjust gain

   • Right-click on ADF image → click Auto Gain

   FIXME: add image showing auto gain result

9. Stop viewing

   • Click View again to stop live scanning

10. Capture line scan (1D EELS)

    • Click Capture and draw a line across the region of interest

      

    • Go to EELS → User Mode

      

    • Go to EELS → Zero Loss → Extract Zero Loss

      

      FIXME: clarify what “extract zero loss” does and expected result

11. Capture area scan (2D EELS)

    • Click Capture and select a rectangular area for 2D spectrum imaging

      

      FIXME: add steps for analyzing 2D EELS data, expected output

Part 4: End session

Follow the steps in End session.

Changelog

• Dec 18, 2024 - initial rough draft by Guoliang Hu and @bobleesj

EDS

Caution

VERY ROUGH DRAFT - @bobleesj and Guoliang Hu took notes and pictures during training. This document will be updated with more detailed steps and images.

TODO:

• Add step-by-step images for TEM mode
• Clarify STEM mode beam settings
• Add screenshots for drift correction setup

This guide covers Energy Dispersive X-ray Spectroscopy (EDS) on the Spectra 300.

Prerequisite: Complete the STEM alignment procedure before starting.

Acronyms:

• EDS - Energy Dispersive X-ray Spectroscopy
• SI - Spectrum Imaging

Part 1: TEM mode EDS

1. Select area

   • Select the area of interest on your sample

2. Open EDS

   • Click on the EDS icon in Velox

     

3. Check experiment log

   • Review the experiment log for acquisition parameters

Part 2: STEM mode EDS

1. Set beam parameters

   • Go to Beam Setting → Probe → click on MF-Y
   • Change convergence angle to 10 mrad (larger area to focus)
   • Increase screen current to 0.4 nA

2. Start spectrum imaging

   • Go to SI (Spectrum Imaging)

   • Click on the rectangle selection tool

   • Select an area on the sample

     

   • For nanoscale resolution, choose 20 microsecond dwell time

3. Set drift correction

   • Click on Drift Area to enable drift correction

   FIXME: add image for drift area selection

4. Adjust parameters

   • Use Object Properties to change acquisition parameters

5. Acquire data

   • Wait until pixel count stabilizes (watch for 8 pixel indicator)

     

   • Stop recording when complete

Part 3: Data processing

1. Draw line profile

   • Go to Processing to draw a line profile across features

     

2. Select arbitrary area

   • You can also select an arbitrary area for analysis

3. Atomic mapping

   • For atomic resolution mapping, use Average or Gaussian filtering

     

   • Use highest frequency and edge smoothing settings to obtain atomic resolution

Part 4: End session

Follow the steps in End session.

Changelog

• Dec 18, 2025 - initial rough draft by Guoliang Hu and @bobleesj

Tomography

TODO: Add tomography tutorial content

This guide covers electron tomography on the Spectra 300.

Changelog

• Dec 17, 2025 - Add placeholder by @bobleesj

Ptychography

For the experiment, record the global meta data:

• keV 300 keV
• convergence angle
• spot size

For region of interest, record the following

• Defocus (nm)
• Mag (Mx)
• Scan steps
• dwell time (μm)
• Camera length
• Data collection notes
• Mono
• Current on flucam: 75 pA
• Note (interesting shape? zone axis?)

Random notes:

• Why is it hard to find zone axis in nanoparticles? Small particles (1 - 10 nm)

PED

TODO: Add PED tutorial content

This guide covers Precession Electron Diffraction (PED) on the Spectra 300.

Changelog

• Dec 17, 2025 - Add placeholder

Share Your Procedure

We want to bring electron microscopy techniques and SOPs together across institutions.

Why contribute?

• Seamless experiments - Researchers traveling between facilities can quickly get up to speed on different microscopes
• Reduced friction - Standardized visual guides reduce training time and errors
• Community knowledge - Your expertise helps the broader EM community

What we’re looking for

• Step-by-step operating procedures for your microscope
• Visual guides with screenshots or photos
• Tips and tricks from experienced operators
• Troubleshooting guides

How to contribute

We can help with writing, formatting, and organizing your content. You don’t need to be a programmer!

1. Prepare your notes - Word docs, Google Docs, handwritten notes, photos from your phone - anything works
2. Reach out - Contact @bobleesj or open an issue on the GitHub repo
3. We’ll handle the rest - We’ll format your content and add it to the site under your institution’s section

Institutions we’d love to hear from

• National labs
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Your procedures will be credited to you and your institution.

Changelog

• Dec 18, 2025 - Page created

Session notes

by Sangjoon Bob Lee

This is a working scratchpad for raw notes taken during microscopy training visits and sessions. Notes here capture practical tips, questions, and observations from hands-on time at the instrument. Over time, useful content gets refined and incorporated into the proper guide sections.

Experiment notes

Gold standard SOP practice: Mar 14, 2026

How to find the ronchigram again?

• Move the joystick around and watch for a small screen current change.
• Then use Diffraction Shift and Focus alignment to find the beam. You will see a faint bit of light.

Remaining questions

• When do you run Stigmator? During real space alignment?
• For C1A1, during the first run, what are the target values? I started with C1 6 nm, A1 27 nm, B2 928 nm. My notes say C1 < 1 nm and A1 < 3 nm. Add target values to the table.
• When do you do manual tuning? With real samples?
• Add a table at the beginning with the target values for aberration corrections.

Open questions

• Ideal Velox play setting? 1024x1024 and 500 ns for gold standard sample.
• When to change condenser stig?
• Camera length and ronchigram size? Proportional: 91 mm to 115 mm, ronchigram size also increases.
• When do you adjust Condenser?

Lessons learned

• Beam condition from FFT: 4-6 rings, each ring with discrete peaks, ~70 pm. Further rings (bigger k) correspond to sharper features resolved in real space.
• Beam condition from Probe corrector: flatness around the “green” aberration surface is the key.
• Overfocus means focal point above the sample; underfocus means below it.
• Defocus change DP? Barely, but real-space probe size is changed (needed for ptycho overlaps).
• Defocus on BF? Expect it to get worse. Ptycho will perform better.
• Focus knob to sharpen? Minimize it. Don’t add degrees of freedom. 20 nm max, use stage piezo and knobs.
• Finding ptycho defocus step size: use ronchigram shadow image to determine feature size variations. Step size of 1 nm is too small.
• Why drift? Inserting the holder itself induces aberrations. After stage movement, find ROI, then wait ~5 min for mechanical stabilization.

Extra notes on aberrations

• Practice getting atomic imaging without Sherpa. Example: Samsung sample, too beam-sensitive for Sherpa.
• Tableau with A5 selected measures up to 5th order aberrations. The following screenshot shows a Tableau measurement result with A5 enabled.

Manual aberration correction without Sherpa (beam-sensitive samples)

• Figure out the full workflow for correcting aberrations without Sherpa.
• Where do you run C1A1 if you can’t expose the sample?
• How do you manually adjust A1 and B2?
• What does “good” look like without Sherpa? Define criteria for the ronchigram, FFT, and probe shape.
• What is the minimum correction quality needed for atomic resolution?

Action steps for future practice sessions

• Try going back to TEM and STEM, confirm aberrations getting worse.
• Try going to LM and then back to regular STEM, confirm resolution getting worse.
• Try correcting the probe without Sherpa, in the case of beam sensitivity.

Cobalt oxide nanoparticles STEM — Mar 3, 2026

It was my first time staying in STEM mode and find samples after STEM probe correction and loading my own sample. The following notes were taken in my attempt to find the feature of interest right after the sample was loaded.

Finding the sample after the sample is loaded

• Go to 5,000× magnification. If there is no beam, it means the beam is blocked on the grid. Move the stage around with the joystick.
• Move the stage until the screen current increases to about 0.150 nA. At this point, the beam has been found. Notice the Kikuchi bands: this is a good clue that you are in a good starting place.

• Increase the magnification to 20,000× or higher. The features will still look blurry since we are not yet at the correct focus.
• Press the Z-axis up and down until the Ronchigram blow-up point appears. Adjust z-axis from 5kx to 20kx to find the blow-up point.

• After the blow-up point, you will see features. In this session, cubes were found after the Ronchigram blow-up point.

• Stay at the blow-up point. We are now at the eucentric height.
• Turn on the HAADF camera in Velox to observe those features.

Getting sharper features after you’ve found them

• Use the stage piezo to move the sample around to ensure you have the sharpest features.
• Use Sharpa to correct C1A1. You do not need to correct B2A2.
• Take the image as usual.

Notes on aberration correctinos

MAPED experience at NCEM, Mar 2, 2026

I had a chance to join TEAM MAPED session at NCEM with Stephanie Ribet and Henry Bell.

General STEM notes for aberration

• LM mode warning: In TEAM, LM mode isn’t used generally. It turns off the aberration corrector, a set of multipole electromagnetic lenses (hexapoles, octupoles) that correct for spherical aberration. When switched back on, the corrector needs hours to restabilize both thermally (coils heat up, causing alignment drift from thermal expansion) and electromagnetically (currents must settle to precise values).
• aberration knobs: in the probe corrector software, manually fix and it can be faster since software can take a while. For example, Correct A1 (twofold astigmatism) and B2 (axial coma) by hand. (@bobleesj, verifiy this)
• Use the stigmator button on the hand panel, it makes the beam round.
• C2 adjust is used to make the beam concentric, by alinigng C2 aperture.
• Rotation center: don’t care about the edges. Use a magnified image to see whether the features are pulsing out of the page.

Sample loading and vacumm

• After loading a sample, watch PPL. It should go down to low 10⁻³ or 10⁻⁴.
• Octagon must be below 10 after sample loading.

Finding sample ROIs

• In Spectra, you can switch between TEM and STEM modes and it’s stable. On TEAM, this is not the case, so it’s better to use STEM at 5k mag to navigate and find samples.
• Use stage double-click to move around.

Zone axis

• Use alpha and beta on the hand panel to get an approximation, then go to Stage, flap out, and use alpha and beta for fine adjustment.
• Feel free to use camera length to make it easier to see. Ensure the ronchigram is symmetric.

Convergence angle

• Change the convergence angle by changing the aperture.
• C2 for 70 µm aperture gives ~9 mrad max. For higher convergence, use another aperture.
• If you change the C2 aperture, the software may still display the old value (e.g., “20”) because it doesn’t know how to get to the new position. Click “Adjust” to move to the new aperture where it has the intended aperature like 10 micrometer bull’s eye aperture.
• Then move C2 to ~30 (instead of 1,000) to block out other apertures.

Arina at NCEM

• HAADF must be out before you insert Arina. Verify on Digital Micrograph.
• Shutdown order: voltage can be turned off from the software. No need to physically turn it off unlike at SNSF.

Colume valve must be closed even during a quick break

• Always set column valve off for lunch. It does not affect aberrations.

Modify current

• To gain more current, go to Focus and Shift under the Mono tab. This controls the monochromator lens excitation, which determines how tightly the beam is focused at the energy-selecting slit. Lowering focus means a less tight crossover at the slit, so more electrons pass through.

Descan pivot point

• TODO: needs definition and procedure.

TODOs

• Investigate the effect of descan when you integrate or sum across k-space

Reconstruction notes

Ptychography reconstruction basics — Mar 9, 2026

Notes from Arthur on reconstructing data collected from ARINA detector at Stanford.

• Sign convention: in quantem, C10 > 0 means underfocus. The beam focal point is below the sample, hence negative defocus.
• Aberrations: SSB is somewhat an “eye test.” One may use the aberrations from SSB or not. There are many degrees of freedom: batch size, the “dose” step size (finer can be better), probe size, center of mass/transpose, and initial aberrations.
• Cropping strategy: in real space, it’s fine to crop, encouraged since faster. In k space, we generally don’t want to crop since we lose the max scattering angle, i.e. we lose fine details in real space.
• Probe: aberrations should be identical across all scan regions in theory. However, for ptycho-tomo, defocus will change with tilt.
• CNN reconstruction: reconstruction weight is different between reconstructions since these are weights being trained.
• Memory: It’s hard to manage memory well in Jupyter notebook but it’s something we can work on.
• Virus samples: there is no zone axis, so we can’t do atomic resolution.
• Descan: the beam is tilted from the source and then tilted back after a short travel perpendicular to the sample. During this second tilt, instability can be introduced and the diffraction pattern isn’t perfectly aligned.
• Mixed probe: mixed probe is good and orthogonality is imposed, so probes should look different from each other.