sample preparation for the three-dimensional reconstruction of pancreatic cancer cell mitochondria

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

Mitochondria are one of the most important organelles in the cell. They serve as the central hub of cellular bioenergetics and metabolism1,2 and play a critical role in cancer3. Pancreatic cancer (PC) is one of the most difficult cancers4 to treat due to its rapid spread and high mortality rate. Mitochondrial dysfunction, which is mainly caused by changes in the mitochondrial morphology3,5,6,7, has been linked to the disease mechanisms underlying PC8. Mitochondria are also highly dynamic, which is reflected by the frequent and dynamic changes in their network connectivity and cristae structure9. The reshaping of the cristae structure can directly impact the mitochondrial function and cellular state10,11, which are significantly altered during tumor cell growth, metastasis, and tumor microenvironment changes12,13.
In recent years, scientists have studied this organelle using EM observation14,15,16,17; for example, researchers have analyzed the mitochondrial dynamics using 3D reconstruction techniques6,7,18,19. The general concept and method for the 3D reconstruction of electron microscopy images were formally established as early as 196820 and involved combining electron microscopy, electron diffraction, and computer image processing to reconstruct the T4 phage tail. Up until now, electron microscopy 3D imaging technology has made significant advancements in terms of the image resolution21, degree of automation22, and processing volume23 and has been used on an increasingly broad scale in biological research, from the tissue level to the organelle ultrastructure level at the nanometer scale24. In recent years, electron microscopy 3D imaging has also become a promising technology for a wide range of applications25,26,27.
The growing attention on mitochondrial cristae particularly illustrates the essential requirements for ultrastructural volume imaging. Transmission electron microscopy (TEM) has been used to visualize samples collected on a copper grid (400 mesh)28, with the electron beam passing through the section. However, due to the limited range of the copper grid, it is impossible to fully image continuous slices of the same sample29. This complicates the study of target structures during TEM imaging. Additionally, TEM relies on time-consuming and error-prone manual tasks, including cutting and collecting multiple slices and imaging them sequentially21, so it is not adapted for ultrastructural reconstructions of large-volume samples23. At present, the high-resolution reconstruction of large-volume sample imaging is achieved through the use of specialized equipment, such as the TEM camera array (TEMCA)30 or two second-generation TEMCA systems (TEMCA2)31, which enable automated high-throughput imaging in a short time. However, this type of imaging does not have the advantage of being easy to obtain and universal due to the requirement for customized equipment.
Compared with TEM, the method for automatically generating thousands of serial volumetric images for large areas based on SEM32,33 enhances the efficiency and reliability of serial imaging and delivers higher z-resolutions34. For example, serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) have both made it possible to achieve the 3D reconstruction of ultrastructure with high speed, efficiency, and resolution35,36. However, it is inevitable that the block surface is mechanically shaved off by the diamond knife of the SBF-SEM or by milling with the focused ion beam of FIB-SEM33,37. Due to the destructiveness of the two methods to the samples, it is not possible to reconstruct the same target structure again for further analysis38,39,40. In addition, few studies have attempted to reconstruct the 3D organelle ultrastructure of cancer cells using EM to observe pathological changes12. For these reasons, in order to further elucidate the pathological mechanisms of cancer cells, such as pancreatic cancer cells, we propose a novel technology for the 3D reconstruction of serial section images using an ultramicrotome and a field emission scanning electron microscope (FE-SEM) to analyze the mitochondrial ultrastructure at the cristae level; with this technology, high-resolution data can be acquired using an efficient and accessible method. The serial ultrathin sections made using an ultramicrotome can be semi-permanently stored in a grid case and reimaged multiple times, even after several years41. FE-SEM is highly valued as a tool in various research fields due to its ability to provide high-resolution imaging, high magnification, and versatility42. In an attempt to display the fine structure of organelles in 3D, the technique for producing serial 2D image stacks with useful resolution using back-scattered electrons produced by FE-SEM43,44 can also be used to achieve high-throughput and multi-scale imaging of target regions or their associated structures without special equipment45. The generation of charge artifacts directly affects the quality of the acquired images, so it is particularly important to keep the dwell time short.
Thus, the present study elaborates on the experimental procedures employed in this SEM technique to reconstruct the 3D structure of mitochondrial cristae46. Specifically, we show the process developed to achieve the semi-automatic segmentation of mitochondrial regions and digitize the 3D reconstruction using Amira software, which also involves making slice samples using the conventional OTO specimen preparation method44,47, completing the section collection using ultramicrotome slicing, and acquiring sequential 2D data by FE-SEM.

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

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 structure at the subcellular nano-scale level, and establishing the link between organelle morphology and the cancer pathological mechanism. This technology combines traditional electron microscopy 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 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 observation.

Watch this Scientific Journal Video about Sample Preparation for the Three-Dimensional Reconstruction of Pancreatic Cancer Cell Mitochondria at JoVE.com

Sample Preparation for the Three-Dimensional Reconstruction of Pancreatic Cancer Cell Mitochondria  

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 2 minutes and discard the supernatant. Fix the cells by adding 1 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 to 1 and incubate at 4 degrees Celsius for 1 hour.Centrifuge and rinse the samples twice with 0.1 molar PBS and once with double distilled water. Then add 1 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 4 times with double distilled water.Next, add 50 microliters of 1%osmium tetroxide and allow it to be fixed at room temperature for 1 hour. Centrifuge again and rinse the samples 4 times with double distilled water. Add 1 milliliter of 2%uranyl acetate and allow it to stain at 4 degrees Celsius overnight.After rinsing the samples with double distilled water add 1 milliliter of Walton solution and incubate at 60 degrees Celsius for one hour. After incubation rinse the cells 4 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 to 1, 1 to 1, and 1 to 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 sliced strips of 70 nanometers thickness on the hydrolyzed silicon wafers and dry them in a 60 degrees Celsius oven for 10 minutes.

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Research

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Cancer Research

159 visualizações.  School of Pharmaceutical Science of Zhejiang Chinese Medical University. Para começar a inocular 2 milhões de células pan02 em 12 mililitros de DMEM, e incubar a 37 graus Celsius e 95% de umidade, 5% de dióxido de carbono e 35% de ar. Após 48 horas, centrifugar a cultura a 28 g por 2 minutos e descartar o sobrenadante. Fixe as células adicionando 1 mililitro de glutaraldeído a 2,5% nos tubos de microcentrífuga de 1,5 mililitro durante a noite a 4 graus Celsius.No dia seguinte, centrifugar as amostras a 1006 g por cinco minutos e aspirar o fixador an...