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Published on: May 4, 2016
The Use of Correlative Light-Electron Microscopy (CLEM) to Study PINK1/Parkin-Mediated Mitophagy
Chieko Kishi-Itakura1, Folma Buss2
1Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK. ci227@cam.ac.uk.
Abstract:
In this chapter we describe the use of correlative light-electron microscopy (CLEM) to study, in cultured cells, the turnover of damaged mitochondria by PINK1/Parkin-dependent mitophagy. CLEM combines the advantages of light microscopy, which allows to image and rapidly screen a large number of the cells, while electron microscopy provides high-resolution imaging of these selected cells and a detailed structural analysis of their cellular organelles. We describe in detail how to prepare the cell cultures for optimum preservation of their cellular ultrastructure for CLEM using the most suitable buffers, fixatives, and embedding resins. These protocols are applicable for detailed ultrastructural analysis in a wide variety of organisms and cells, ranging from prokaryotic bacteria to mammalian cells.
Insights
Correlative light-electron microscopy (CLEM) enables detailed study of mitophagy, the process by which cells clear damaged mitochondria. This chapter details CLEM protocols for ultrastructural analysis in diverse cell types.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Mitochondrial Biology
Background:
- Mitophagy is crucial for cellular health, involving the removal of damaged mitochondria.
- The PINK1/Parkin pathway is a key regulator of mitophagy.
- Understanding mitophagy dynamics requires high-resolution imaging techniques.
Purpose of the Study:
- To describe the application of correlative light-electron microscopy (CLEM) for studying mitophagy.
- To provide detailed protocols for preparing cell cultures for CLEM.
- To enable ultrastructural analysis of mitochondrial turnover in cultured cells.
Main Methods:
- Correlative light-electron microscopy (CLEM) combining live-cell imaging and high-resolution electron microscopy.
- Cell culture preparation optimized for ultrastructural preservation.
- Detailed protocols for fixation, embedding, and resin selection for CLEM.
Main Results:
- CLEM allows rapid screening of numerous cells using light microscopy.
- Electron microscopy provides detailed structural insights into selected cells and organelles.
- Optimized protocols ensure high-quality ultrastructural preservation for CLEM analysis.
Conclusions:
- CLEM is a powerful technique for investigating PINK1/Parkin-dependent mitophagy.
- The described protocols are versatile and applicable across various organisms and cell types.
- This chapter provides a valuable resource for researchers studying mitochondrial dynamics and cellular quality control.
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