The overall goal of this procedure is to obtain a three-dimensional reconstruction with depth of field recovery of a thick biological specimen using convergent beam scanning transmission electron tomography. This method can answer key questions in the field of structural biology about electron tomography of thick biological specimens. The main advantage of this technique is that images can be acquired in convergent bit mode and the data processing can be performed on most software dedicated to tomography.

To begin preparing cell culture sample, centrifuge the sample at 5000 x g for five minutes. Use these centrifugation parameters throughout sample preparation. Discard the supernatant and resuspend the cells in one milliliter of PBS enriched with 4%paraformaldehyde and 2%gluteraldehyde.

Incubate the suspension for 30 minutes at room temperature. Centrifuge the mixture, discard the supernatant, and wash the pellet three times with one milliliter portions of PBS. Using the same incubation conditions and washing procedure, incubate the pellet in one milliliter of PBS, enriched with 1%osmium tetroxide, and in one milliliter of PBS enriched with 2%uranyl acetate.

Next, cool the sample to four degrees Celsius. Dehydrate the pellet by successive incubations in increasingly concentrated ethanol solutions. Maintaining the sample temperature of four degrees Celsius throughout incubation, centrifugation, and washing.

Resuspend the dehydrated pellet in one milliliter of pure ethanol, and incubate overnight at 4 degrees Celsius. Then, allow the sample to warm to room temperature, and centrifuge the sample. Incubate the pellet in increasingly concentrated epoxy resin solutions at room temperature.

After the final wash, resuspend the pellet in one milliliter of pure epoxy resin, and incubate overnight at room temperature. Centrifuge the sample, and remove the supernatant, and then resuspend the pellet in 200 microliters of epoxy resin and hardener. Transfer the suspension into the capsule.

Incubate the plastic capsule at 60 degrees Celsius for 48 hours to polymerize the resin. Remove the capsule from the incubator and verify resin polymerization. Add a layer of epoxy resin and hardener over the embedded sample and incubate the capsule again under the same conditions.

Mount the sample embedded in resin upside down in a specimen holder. Securely fix the sample on the trimming block of an ultramicrotome. Cut the plastic capsule away from the sample in the resin.

Shape the exposed resin into a pyramid. Then transfer the capsule and specimen holder to the moving arm of the ultramicrotome. Install a trimming knife and guided by a microscope, refine the pyramid shape.

Replace the trimming knife with a histology knife. Under the microscope, adjust the capsule pitch angle so the pyramid is perpendicular to the knife edge. Set the cutting speed and section the sample.

After sectioning the sample, transfer the chosen section to a treated copper grid over filter paper. Allow the sample to dry, and then load the sample into the electron microscope. To design the through focal tilt series, first calculate the electron beam depth of field and the maximal apparent thickness of the sample.

Based on these values, select a focus interval and determine the number of focal steps needed to image the entire sample at the maximal apparent thickness. Then, at low magnification, locate the region of interest within the sample. Adjust the eucentric height so that the ROI does not appear to move when the sample is rotated.

Verify that the ROI remains visible throughout the tilt range. Fill in the through focal tilt series parameters for automated image collection and acquire the images. Import the image series into an image processing program, and align the images collected at the same tilt angle in a pyramidal hierarchy.

Find the alignment at each tilt angle by stacking the aligned images. Browse through the stack to ensure that only the focused region moves from image to image and realign the images as needed. Once alignment is verified throughout the sample, merge the in-focus information at the high settings to yield the series with a single image per tilt angle.

Perform find alignment of the tilt series based on local minima or fiducial markers. Perform a 3-D reconstruction and inspect the resulting image. If the reconstruction appears blurred or deformed, realign the original image series.

A sample of t. brucei was imaged and analyzed using this method. At a zero degree tilt angle, several details of the flagellar pocket are visible.

At the higher tilt angle of 70 degrees, only a portion of each image collected in the through focal series is in focus. This position shifts throughout the series. By merging the in-focus sections, a single image with a larger in-focus zone is produced.

This effect is even more pronounced when the high frequency areas of the through focal tilt series are highlighted. The high frequency information spans most of the combined image, indicating that the through focal tilt series has been processed well. Once mastered, the image processing part can be done in around three to four hours, depending on the chosen alignment and reconstruction parameters.

While attempting this procedure, it's important to remember that the quality of the final reconstruction greatly depends on the quality of the previous alignments tests. After watching this video, you should have a good understanding of how to perform the collection and the image processing of a through focal tilt series.