Cut a conductive carbon tape and stick it between the silicon wafers mounted with pancreatic cell sections and SEM specimen stage. Next, set the FESEM accelerating voltage to two kilovolts with a working distance of four millimeters. In the top menu bar, click on the HL icon.
Then, at low magnification, orient the first section of the target slice strips, and, again, click on the HL icon to switch to the high magnification mode. Collect an appropriate image for the structure of interest by adjusting the image brightness, contrast, and magnification. Next, select Project View, then Open Data, and import the image files to be analyzed into the software.
Align the pictures by clicking on Align Slices and Edit. Then, in the lower left menu bar, adjust the Intensity Range value by modifying the image transparency. Select the Extract Sub-Volume module, and click the aligned data sets to fit the size of the overlapping portion of the whole stack.
Next, under the segmentation sub-section, select Resample, then Segmentation, and click Save. Then, choose the threshold of the Magic Wand, and the size of the Brush Tool, to select the correct range. From the Selection menu, click on the Add icon to add the selected area.
After completing the regional segmentation, generate an image file following the best results for the size of the object and picture resolution. Click on Crop Editor, and in the Virtual Slider box, enter the number six. Next, from the left dropdown menu, right-click on the gray area under the Project sub-section and select Generate Surface, then Create And Apply.
In the generated file, using the Surface View module, create the surface structure and 3D representation. The 2D FESEM images reveal that in the control group, mitochondria were evenly distributed with regular cristae structures. In contrast, the RSL3 group showed shrunk mitochondria with increased membrane density and vague cristae structures.
However, not all mitochondrial cristae degenerated in the RSL3 group as some remained intact. Compared to the RSL3 group, the inhibitor group had mostly intact cristae structures with only a few not apparent. The 3D images of the control group provided a more accurate view of the mitochondria and their cristae structures.
The RSL3 group 3D images showed irregular and vacuolated mitochondria, while the inhibitor group displayed diverse cristae shapes. The quantification of mitochondrial alterations showed that the RSL3 group had significantly decreased mitochondrial length and volume, compared to the control group, while the inhibitor group showed intermediate results. RSL3 also caused mitochondrial dysfunction and altered cellular metabolism by changing mitochondrial morphology and triggering ferroptosis.
