The FEI Titan 80-300 TEM is a high-resolution transmission electron microscope (TEM) equipped with a field emission gun and a corrector for the spherical aberration (CS) of the imaging lens system. Digital images are acquired with a Gatan 2k x 2k slow-scan charged coupled device camera. The instrument is used for the investigation of a wide range of solid state phenomena on the atomic scale, but mostly high-resolution transmission electron microscopy (HRTEM) utilizing negative spherical-aberration imaging (NCSI) technique. The machine can be adapted to utilize low temperature holders and external electric/magnetic fields. It is moderately suited and occasionally used for diffraction studies (text adapted from ER-C website).

This section lists all the steps necessary to start a diffraction experiment on the Titan from its default configuration. The information in the parenthesis following the instruction specifies in the part of FEI GUI or hand panel where the control is located.

Load default configuration

• Open column valves (Vacuum » Vacuum)

• Load 300kV TEM C3-off registry to set the basic alignments (Vacuum » FEG Registries)

• Select and apply available 300kV alignments (Align » Alignments » File)

• Set the highest available gun lens setting of 8 (Vacuum » FEG Control)

• Set one of the higher available spot size settings, at least 6 compared to the default of 3 (Column » Beam Settings)

The highest available gun lens and spot size settings warrant the lowest intensity of the incident beam. This lowers the amount of damage dealt to the sample and the detector while still sufficing for the diffraction method. Setting FEG registry also automatically resets the apartures and removes the camera from the column. In order to bring it back at any time, select "Camera » CCD/TV Camera » Insert" or start any acquisition from the Digital Micrograph window.

Align the beam

After loading the registry and setting all the initial alignments, the beam should be visible at one of the lowest magnification settings e.g. SA 3400x. If the beam is not visible on the fluorescent screens, it may be shadowed by a part of the sample grid (move joystick) or blanked (Camera » CCD/TV Camera » Blank). In case the beam can still not be found, request assistance of the laboratory staff.

Continue to refine the basic loaded alignment:

• Set a magnification level for the diffraction experiment e.g. SA 11500x (hand panel)

• Navigate to the direct alignments (Align » Direct Alignments)

• Throughout the alignments, keep the beam focused using the "Intensity" knob.

To start the following alignments, select them on the list. To finish each alignment, switch to another one or click "Done". Preferably, perform these alignments on the little fluorescent screen, lifted using the lever left of the viewing chamber.

• Calibrate the beam tilt pivot points X and Y by overlapping the split beam components using the Multifunction X/Y knobs and correcting for the beam shift as needed using the track ball

• Calibrate the beam shift by moving the beam to the middle of the screen using the track ball

The rotation center calibration may be performed but is difficult to perform using the tools provided and as such typically not worth it.

Switch to the lowest available condenser i.e. 50 (Column » Apertures » Condenser 2). After performing listed alignments, the beam may be a bit elliptical i.e. astigmatic. Astigmatism of the beam should not be judged on the little screen as it is tilted. To correct for it, switch to the large fluorescent screen or the camera and:

• Navigate to the stigmator and select "condenser" (Camera » Stigmator)

• Use Multifunction knobs to make the beam circular

• Switch the stigmator back to "None"

Stigmator correction might require repeated calibration of the beam shift. If time allows, it may be a good idea to repeat all manual alignments altogether to warrant high quality.

Identify the sample

Once the beam is circular and spreads concentrically, proceed to look for a fine crystal for measurements. Once a suitable i.e. clear, uniform, and semi-transparent candidate is found, determine the correct eucentric height and save the stage position:

• Navigate to and start the wobbler with low angle (Stage » Control » Alpha Wobbler)

• At increasing wobbling angles, minimize sample movement using the hand panel Z-height buttons

• Once the crystal stops drifting with alpha, add or update the stage position (Stage » Stage 2)

The stage tilt has an upper practical limit of 50 degrees, but to keep the drift managable it is advised to stay within 20 to 30 degrees. There is some backlash on the alpha angle, which at the time of writing is not accounted for in the software.

In order to identify the beam size:

• In the Digital Micrograph GMS GUI window, create any view and start any acquisition

• Once an image is collected, from the toolbar select a "profile" tool

• Click on both sides of the displayed beam

• Click the edges of the new beam profile histogram to calculate the spacing between them

Long-term calibrations have been already done and saved in the %appdata% directory. They should need to be repeated. However, some experiments and functionalities require a recent beam shift calibration that translates between `beamshift` settings and detector pixel coordinates. This calibration, if needed, should be stored in the calib folder of the current experiment directory.

In order to manually prepare a new beam shift calibration (automatic process was semi-successful):

• Prepare the microscope configuration: set desired magnification and spot size, move the beam to the camera center, and focus it as much as possible 

• Create a new directory `calib` inside your current experiment directory (e.g. `D:\Titan_Data\DanielT\2026-01-06\calib`)

• Open a command line window and navigate to the newly-created directory

• Activate the virtual environment: `conda activate Instamatic`

• Start `instamatic.calibrate_beamshift` and confirm the calibration.

It is preferrable but not necessary for the beam to be visible in all the points on the detector. After the calibration is started, the beam will move to `gridsize**2` individual positions on a square grid, spaced apart by `stepsize` beam shift setting values each, collect, and save an image in each. Using cross-correlation between the images, the program will calculate the transformation matrix that will be saved in a newly created `calib_beamshift.yaml` file.

In a simplest experimental scenario, when no tracking is required and stills in equal intervals is all that is needed, a simple RED experiment will do the trick. This experiment can be run using two different experimental frames, "RED" and "FastADT", the former being the simpler one. In general, Instamatic experiment functionality is split across these frames, typically each of which has been prepared by a different author for unique purpose.

The "FastADT" has been designed in 2025 by the author as a functional replacement of the analogous protocol implemented as RATS: an iTEM plugin created by Yaşar Krysiak, Sergi Plana-Ruiz, and Lukáš Palatinus. It is capable of collecting RED, PED, and cRED data with a priori tracking. Compared to other single-crystal frames of Instamatic, it is robust and transferable but may require additional calibrations to run some of the cases.

To start a "FastADT" experiment, select diffraction and tracking settings. Additionally, manually select and "store" the beam settings for taking "image", "tracking" crystal, and collecting "diffraction". These settings will be automatically recalled as needed during the experiment, and can be recalled manually using the "restore" buttons. Additionally, "beam blank" can be controlled using a dedicated button. Once everything is set, click "start" and follow the instructions on the screen (if any).