Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

Area of Science:

• Microscopy and Imaging
• Cell Biology
• Virology

Background:

• Accurate localization of nucleic acids is crucial for understanding viral infection mechanisms.
• Traditional electron microscopy techniques can be limited in elemental mapping and prone to artifacts.

Purpose of the Study:

• To develop and validate a robust method for mapping nucleic acids in virus-infected cells using Energy Filtered Transmission Electron Microscopy (EFTEM).
• To accurately determine the RNA content of coronavirus particles within infected cells.

Main Methods:

• Utilized EFTEM with phosphorus mapping (P-maps) on unstained thin sections of virus-infected swine testis (ST) cells.
• Applied N-windows Egerton model for background subtraction and optimized sample preparation to minimize artifacts.
• Employed a new software for interactive image processing and Multivariate Statistical Analysis for signal enhancement and noise reduction.

Main Results:

• Generated high-quality P-maps with improved signal-to-noise ratio (SNR) by effectively separating characteristic signals from noise.
• Successfully localized nucleic acids in different cellular compartments, distinguishing between high-concentration areas (chromatin, ribosomes) and low-concentration areas (mitochondria).
• Quantified the RNA content of maturation intermediate coronavirus particles within infected cells.

Conclusions:

• The developed EFTEM-based methodology provides accurate nucleic acid localization in unstained biological samples.
• This technique is effective for studying viral RNA distribution and content in infected cells, offering insights into viral replication and maturation.