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A protocol for cryogenic volumetric imaging using serial plasma FIB/SEM.

Maud Dumoux1, Jake L R Smith2, Thomas Glen1

  • 1Structural Biology, The Rosalind Franklin Institute, Harwell Science & Innovation Campus, Didcot, United Kingdom.

Methods in Cell Biology
|July 14, 2023
PubMed
Summary

This study details a protocol for cryogenic volumetric imaging using serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM). The technique offers near-native imaging of biological samples at high resolution for correlative microscopy.

Keywords:
CryogenicPlasma FIB/SEMSlice and viewVolume EM

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Area of Science:

  • Life Sciences
  • Microscopy
  • Biotechnology

Background:

  • Correlative volume electron microscopy (vEM) techniques are crucial for high-resolution imaging of biological structures.
  • Serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM) is an emerging vEM method for visualizing unstained, cryogenically preserved samples.
  • Achieving optimal imaging parameters is essential for reducing artifacts and enabling downstream analysis.

Purpose of the Study:

  • To provide a detailed, hardware-agnostic protocol for optimizing serial pFIB/SEM parameters.
  • To minimize imaging artifacts and facilitate computational processing and analysis of volumetric data.
  • To enable near-native state imaging for microscale, mesoscale, and nanoscale correlative imaging.

Main Methods:

  • Development of a protocol for serial pFIB/SEM focusing on optimization of FIB and SEM parameters.
  • Emphasis on the purpose of each step to ensure hardware and software agnosticism.
  • Guidance for achieving ∼20-50nm resolution and a field of view of ∼10-30μm.

Main Results:

  • A comprehensive protocol for serial pFIB/SEM has been established.
  • The protocol addresses the optimization of imaging parameters to reduce artifacts.
  • The method allows for near-native state imaging of cells and tissues.

Conclusions:

  • The developed protocol serves as a valuable resource for researchers using serial pFIB/SEM.
  • Optimization of parameters is key to successful artifact reduction and data analysis.
  • This technique advances correlative imaging capabilities across multiple scales.