Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

Area of Science:

• Electron microscopy
• Biological imaging
• Cellular ultrastructure

Background:

• Back-scattered electron (BSE) imaging is crucial for biological specimens.
• Low accelerating voltage and beam current are desirable for minimizing sample damage.
• Traditional methods face challenges in achieving high contrast and resolution simultaneously.

Purpose of the Study:

• To evaluate the effectiveness of beam deceleration (BD) for back-scattered electron (BSE) imaging.
• To improve compositional contrast and resolution in resin-embedded biological samples.
• To develop a solution for stage tilting-induced astigmatism in focused ion beam/scanning electron microscopy (FIB/SEM).

Main Methods:

• Applied beam deceleration (BD) with 0-4 kV to BSE imaging of en bloc stained rat hepatocytes.
• Utilized low accelerating voltage (1.5-3 keV) and low beam current (10 pA).
• Designed and tested a novel specimen holder for FIB/SEM applications to mitigate astigmatism.

Main Results:

• BD significantly enhanced compositional contrast and improved resolution at low landing energies.
• BD imaging was effective even at long working distances.
• The novel specimen holder enabled high-resolution (approx. 5 nm) 3D reconstruction of cellular organelles and mitochondrial cristae.

Conclusions:

• Beam deceleration is advantageous for block face imaging of biological materials.
• BD offers improved contrast and resolution under low-voltage, low-beam-current conditions.
• The developed specimen holder facilitates high-quality FIB/SEM analysis of cellular ultrastructure.