Visualization of Organelles In Situ by Cryo-STEM Tomography

TEM tomography is widely used for imaging of cells, but it's very limited in terms of specimen thickness. FIB SEM and soft x-ray tomography can be used for larger samples, albeit at lower resolution. STEM tomography fills this gap perfectly.

STEM tomography provides a look at micron thick samples at a resolution of a few nanometers. The combination with cryogenic preparation lets us look at biological samples and sections in 3D. Review the basics of STEM optics in order to understand image formation and ensure that the service engineer has aligned well the stem and Lomax stem modes, and that the calibration steps look like those in the video.

To begin, load the column alignment file and open the column values. If using a side-entry cryo holder, open the cryo shield and start in the TEM mode. The beam should appear on the screen.

Lower the magnification if the beam does not show up. Bring the microscope to eucentric focus by pressing the button on the control panel. Set the spot size to a convenient value for visualizing the fluorescent screen directly, or with the built-in camera.

Set the microscope to STEM mode and verify that the focus uses the condenser lenses rather than the objective. On the panel, set eucentric focus and go out of diffraction mode for initial adjustments. Ensure the beam is not blank and reduce the magnification until the beam appears on the screen.

Adjust the beam shift to the center and increase the magnification in steps up to 70, 000, while keeping the beam in the center. Then insert the desired condenser aperture, typically 50 micrometers for micro probe mode, and check the aperture centering. While turning the focus knob back and forth, the spot should expand and contract, but remain in place, as if a plane cuts an imaginary vertical hourglass.

If the aperture is not centered, the illumination will shift laterally as if the hourglass were tilted. Bring the beam to focus, press intensity list focus in the alignments tab, or return to eucentric focus. Readjust the beam position to the center and adjust the rotation center.

Now, turn the focus step wheel to the minimum, or one step above, so that the beam pulses gently and ensure it remains stationary as the focus moves up and down. Select the pivot points and bring the two points together with X and Y adjustments. Adjust the condenser stigmators to make the beam round.

Go up and down through focus to optimize. There should be no tendency to elongate in one direction or the other when passing through focus. Normalize the lenses, then increase the magnification progressively to about 240, 000, while using the beam shift to keep the spot centered and repeat the rotation center and pivot point adjustments.

Return to diffraction mode. At this stage, the beam should appear as a uniform disc on the fluorescent screen. The camera length now effectively controls the optical distance to the detector as an x-ray crystallography.

Change it and watch as the disc contracts and enlarges as if the screen location would move toward or away from the specimen. This cone represents the brightfield illumination. To engage STEM mode at high magnification, Start with the brightfield stem detector and adjust the diffraction alignment to center the beam using the desired camera length.

Turn on the brightfield marking on the screen, reduce the camera length to 330, lift the screen and insert the detectors. Start a scan in the microscope software. Use the scope display to assist while adjusting the brightness and contrast settings as described in the manuscript.

Iterate the adjustment several times. Return the microscope to a relatively low magnification in the high magnification register, without entering low magnification mode. Insert the fluorescent screen and note the screen current for reference.

As in TEM, the current can be changed with the gun lens and spot size settings with increasing numbers corresponding to a reduced current. At this point, save a FEG register to facilitate a return to standard values. Go to LMTEM mode in order to see the specimen.

Insert the specimen. Bring the sample to eucentric height. There are several methods to do this.

For example, use the stage wobbler to tilt the grid while moving the specimen height along the Z axis until the image stops shifting laterally. Alternatively, mark some features on the viewing screen and tilt the stage to 10 to 30 degrees. The feature will move laterally.

Adjust the specimen height to bring it back to its original position. Increase the magnification or stage tilt to refine and return the tilt to zero degrees. Now return to STEM mode and insert the STEM detector.

Ensure that enable LM scan is unchecked. Go to the lowest high magnification mode. Tune the condenser astigmatism by the method.

With the beam over a thin sample area, focus on the point where the transmitted beam blows up in between shadow images of the sample on either side. Then adjust the condenser tuning to make the central disc round. This requires some practice, especially for cryogenic samples.

Go back to the 50 micrometer aperture and update the FEG register. Go to LMSTEM mode and continue scanning to find an interesting area. If necessary, readjust the detector brightness and contrast settings roughly at this point.

Press eucentric focus, increase the magnification and refine the focus while scanning using the focus loop provided by the microscope and check the astigmatism on gold beads. Until now, everything is performed in nano probe mode. Notice that inserting a smaller condenser aperture decreases the semi convergence angle, which is helpful for thick samples.

An alternative approach with TFS instruments is to switch to micro probe mode, which leads to a reduced semi convergence angle. Then, adjust camera length in order to have the collection angle three times larger than the convergence angle;i. e, the BF detector area markings on the screen are around three times larger than the beam.

Estimate the dose for recording using an equation. As a rule of thumb, aim from 100 to 150 electrons per square inkstrom for the whole tomograph. Return the stage to a whole and adjust the beam current using spot size and or gun lens settings to reach the desired screen current.

Full low magnification grid map recorded in STEM mode shows the areas with cells of interest. Cells appear partly bright with electrons scattered toward the HAADF detector. Medium resolution map recorded in STEM mode displayed two medium resolution anchor maps.

Zero degrees tilt of a stem tilt series allowed the visualization of the calcium phosphate deposits, cristae and gold fiducial markers of a mitochondrion. Volume rendering of 60 nanometers and 40 nanometer thick sections are shown. Cryo stem tomography, or CSTET, provides a bridge between structural biology and cell biology, as well as a context for super resolution fluorescence.

Without the need for sectioning or lamella preparation, we can see the whole cellular theater in a near native state.