A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

This project aims to review the anti-cancer effect of drugs by changing the ultrastructure at the subcellular nanoscale level and establishing the link between organelle morphology and the cancer pathological mechanism. This technology combines traditional electron macroscopic technology with three dimensional reconstruction technology without special equipment. Compared to other techniques, it can improve imaging efficiency and the reconstruction flexibility and the real life reconstruction of target structures at any time, so it's highly easy and universal.

This technology of three dimensional reconstruction of the continuous ultra-thin slides can repeatedly restore the real the real stereochemical without specific expensive equipment. It can be used for qualitative and quantitative analysis of all organelles at any time. It realizes the easy operation of ultrastructure observation.

To begin, inoculate 2 million Pan02 cells in 12 milliliters of DMEM and incubate at 37 degrees Celsius and 95%humidity, 5%carbon dioxide, and 35%air. After 48 hours, centrifuge the culture at 28 g for two minutes and discard the supernatant. Fix the cells by adding one milliliter of 2.5%glutaraldehyde in the 1.5 milliliter micro centrifuge tubes overnight at 4 degrees Celsius.

The next day, centrifuge the samples at 1006 g for five minutes and aspirate the fixative before rinsing the sample twice with 0.1 molar PBS. Subsequently rinse the samples twice with double distilled water for 10 minutes each. Next, add a 50 microliter solution of 1%osmium tetroxide, and 1.5%potassium ferrocyanide at a ratio of 1:1 and incubate at 4 degrees Celsius for one hour.

Centrifuge and rinse the samples twice with 0.1 molar PBS and once with double distilled water. Then add one milliliter of 1%thiocarbohydrazide and incubate at room temperature for 30 to 60 minutes After incubation, centrifuge and discard the supernatant before rinsing the samples four times with double distilled water. Next, add 50 microliters of 1%osmium tetroxide, and allow it to be fixed at room temperature for one hour.

Centrifuge again and rinse the samples four times with double distilled water. Add one milliliter of 2%uranyl acetate and allow it to stain at four degrees Celsius overnight. After rinsing the samples with double distilled water, add one milliliter of Walton solution and incubate at 60 degrees Celsius for one hour.

After incubation, rinse the cells four times with double distilled water before dehydration in a graded ethanol series for 10 minutes each. Then dehydrate twice in one milliliter of 100%acetone for 10 minutes each. Next, mix 200 microliters of acetone with Pon 812 epoxy resin at a ratio of 3:1, 1:1, and 1:3 and soak the samples in the resin at room temperature for 2, 4, and 4 hours respectively.

After respective incubation, impregnate the samples overnight in 100%Pon 812 epoxy resin. The next day, transfer the specimens into a flat embedding mold and polymerize them in an oven at 60 degrees Celsius for 48 hours. After polymerization, using an ultra microtome collect serially slice strips of 70 nanometers thickness on the hydrolyzed silicon wafers, and dry them in a 60 degrees Celsius oven for 10 minutes.

Cut a conductive carbon tape and stick it between the silicon wafers mounted with pancreatic cell sections and SEM specimen stage. Next, set the FE-SEM 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 strip 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 subsection, 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 subsection 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 FE-SEM images revealed 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.