Published: June 23rd, 2023
This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.
Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.
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 ....
1. Material preparation
During cell culture (Figure 1A), we first divided the pancreatic cancer cells into a control group cultured with complete culture medium, a (1S,3R)-RSL348 (RSL3, a ferroptosis activator, 100 nM) group, and an RSL3 (100 nM) plus ferrostatin-149 (Fer-1, a ferroptosis inhibitor, 100 nM) group. Through the above experimental steps, the scanning electron microscope acquired 38 (Supplementary Figure 1), 43 (Supplementary.......
The method presented here is a useful step-by-step guide for applying the 3D reconstruction technique, which involves applying electron microscopy and image processing technology to the stacking and segmentation of 2D tomographic images generated from serial ultrathin sections. This protocol highlights a limitation of 2D images that can be addressed by 3D visualization of the organelle ultrastructure, which has the advantages of strong reproducibility of the structures at a high-resolution level and higher accuracy. More.......
This research was supported by Natural Science Foundation of Zhejiang Province grants (Z23H290001, LY19H280001); National Natural Science Foundation of China grants (82274364, 81673607, and 81774011); as well as the Public Welfare Research Project of Huzhou Science and Technology Grant (2021GY49, 2018GZ24). We appreciate the great help, technical support, and experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.....
|Dulbecco's modified Eagle’s medium
|Fetal bovine serum
|Field emission scanning electron microscope
|European Collection of Authenticated Cell Cultures
|Phosphate Buffered Saline
|Pon 812 Epoxy resin
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