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

Summary

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

Abstract

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.

Introduction

Mitochondria are one of the most important organelles in the cell. They serve as the central hub of cellular bioenergetics and metabolism^1,2 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 morphology^3,5,6,7, has been linked to the disease mechanisms ....

Protocol

1. Material preparation

1. Culture 2 x 10⁶ Panc02 cells in 12 mL of DMEM medium (10% fetal bovine serum and 100 U/mL of penicillin-streptomycin), and maintain at 37 °C and 95% humidity in an atmosphere of 5% carbon dioxide and 95% air for 48 h.
2. Collect Panc02 cells, centrifuge at 28 x g for 2 min, and then discard the supernatant. Ensure that the sample is of an appropriate size (1 x 10⁷ cells), as otherwise, the following fixation and dehydration steps will not work well.
3. Add 1 mL of 2.5% glutaraldehyde as a fixative to fix the fresh Panc02 cells in the 1.5 mL micro-centrifuge tubes ove....

Results

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 Figu.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(1S,3R)-RSL3	MCE	HY-100218A
Acetone	SIGMA	179124
Amira	Visage Imaging		2020.2
Aspartic acid	MCE	HY-42068
Dulbecco's modified Eagle’s medium	Gibco	11995115
Ethanol	Merck	100983
Ferrostatin-1	MCE	HY-100579
Fetal bovine serum	Gibco	10437010
Field emission scanning electron microscope	HITACHI	SU8010
Glutaraldehyde	Alfa Aesar	A10500.22
Lead nitrate	SANTA CRUZ	sc-211724
Osmium Tetroxide	SANTA CRUZ	sc-206008B
Panc02	European Collection of Authenticated Cell Cultures	98102213
Penicillin-streptomycin	Biosharp	BL505A
Phosphate Buffered Saline	Biosharp	BL302A
Pon 812 Epoxy resin	SPI CHEM	GS02660
Potassium ferrocyanide	Macklin	P816416
Thiocarbohydrazide	Merck	223220
Ultramicrotome	LEICA	EMUC7
Uranyl Acetate	RHAWN	R032929