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