This protocol allows 3D reconstruction of organelles in a simple and robust manner, whilst being accessible to laboratories that do not currently have specialist volume electron microscopes. This technique is extremely flexible, providing excellent X/Y resolution, whilst being able to re-image samples if necessary. It's also compatible with tilt tomography when very high Z resolution is required.
This technique allows interrogations of organelle morphology in inter-organelle context. Therefore, it can be extended to examine any pathological conditions with organelle defects. This approach can provide insight into organelle interactions and cellular trafficking events.
This method can also be applied to other tissues, organoid models, and cell culture systems, in addition to hepatocytes. First-time users may find serial sectioning and pick up challenging without loss. It's recommended to be proficient at regular ultra-thin sectioning before applying this protocol to a precious sample.
Begin by carefully trimming the resin-embedded tissue using a razor blade with the sample locked in the trimming adapter. Transfer the specimen ark together with the chuck and the sample to the specimen arm of the microtome and position the specimen ark so that the ark's travel range runs from top to bottom. Place and lock the diamond knife in the knife holder, ensuring that the knife's cutting angle is set appropriately.
Lock the knife holder into the stage securely. Turn off the stage downlighting, turn on the stage uplighting, cautiously move the knife towards the specimen, continually adjusting the knife's lateral angle, specimen tilt, and specimen rotation by adjusting the relevant knobs until the block face is aligned to the knife's edge. Set the top and bottom of the cutting window of the specimen arm and leave the specimen just below the knife edge.
Fill the knife boat with clean, double-distilled water and ensure the water surface is level with the knife's edge and slightly concave. Then, start cutting sections. Stop the cutting just after the sample has passed the knife's edge.
Use an empty slot grid to estimate the number of sections to collect on each grid. Using an eyelash in each hand, gently break the ribbon into smaller ribbons that can fit in the length of the slot grid. Make a mental note of their relative positions within the sample.
Using a glass applicator rod, hover a drop of chloroform over the sections to flatten them out. Pick up the first empty slot grid with the first numbered forceps and gently dip it in the Triton X-100 and then two times in the distilled water. Use a piece of filter paper to remove the excess water from the forceps'edge.
Using forceps, gently lower approximately 2/3 of the formvar-coated slot grid into the water of the knife boat. Ensure that the formvar side is facing down and the long right hand edge of the slot is at the surface of the water and parallel to the water's edge. Gently waft the grid in the water towards the ribbons so that upon the return stroke, the sections drift towards the grid.
Continue to do this in smaller and smaller wafts, until the right hand edge of the ribbon lines up with the right hand edge of the slot. Then, with the last waft, gently bring the grid up to pick the sections up into the slot grid. Place several pellets of sodium hydroxide under a Petri dish lid to provide a carbon dioxide-free environment.
Then, carefully pipette 40-microliter drops of Reynold's lead citrate on the parafilm, one drop for each grid. Invert each grid onto the lead citrate drop and leave protected by the Petri dish lid for 7 to 10 minutes. While the grids are staining, place five approximately-300-microliter drops of distilled water for each grid on the parafilm.
At the end of the lead citrate incubation, transfer each grid to a droplet of distilled water to wash for one minute without breathing directly on the grids. Repeat this four more times. Use numbered crossover forceps to pick up the first grid.
Touch the edge of the grid to filter paper to wick away most of the water and allow to dry in the forceps for at least 20 minutes. Repeat this for each grid. At low magnification, observe the order, location, and position of the serial sections.
Navigate to the middle section of the series on the grid. Browse the sample and identify a region-of-interest. Then, observe the sample at the desired magnification.
Consider collecting the series at a slightly lower magnification, as sections are often not perfectly aligned, and images may need to be cropped later. Next, take reference images at lower magnifications. This will help to appreciate the context of the region-of-interest, it's rough location at different magnifications in relation to section boundaries, and landmark features within the sample.
Use these to signpost the region-of-interest in other sections. For screen referencing, use reusable adhesive putty, stickers, or a piece of overhead projector paper taped to the screen to place temporary markers on the screen. This will allow routine re-imagining of the same features of the region-of-interest in the center of the image throughout the dataset.
Open the image stack, specify the voxel measurements, and select the Segmentation tab to start segmentation. Click New in the segmentation editor panel to define new objects in the material list. Right click to change the color of the object and double click to rename the object.
For manual segmentation, choose the segmentation tool below the material list and select the default brush tool to highlight the pixels. The electron microscopy image of two opposed hepatocytes is shown here. Higher magnification imaging allows the observation of the morphological details of the different organelles with nanometer resolution.
The representative image shows the segmented version of the same tomogram, trace of the endoplasmic reticulum, mitochondria, and the intermembrane contacts between the ER and the mitochondria of different spaces are shown here. The representative image shows a tilted ortho-slice overlaid with segmentation traces and its relative position within the whole mitochondrion. 3D reconstruction of the segmented organelles and the endoplasmic reticulum-mitochondria contacts at different angles are shown here.
As this protocol is compatible with tilt tomography when very high Z resolution is needed, it helps to resolve small or convoluted structures. This technique is perfect for the initial assessment of the spatial relationship between different organelles, and, therefore, particularly useful in the progressing field of membrane contacts at membrane homeostasis.
