Shaping cryogenic 3D volume imaging by pFIB ion species and milling voltage

Cryogenic plasma focused ion beam-scanning electron microscopy (cryo-pFIB-SEM) enables nanoscale volume imaging of vitrified cells and tissues, but the fidelity of each newly exposed block-face depends on how ions interact with compositionally heterogeneous biological material. Using xenon, argon, oxygen, and nitrogen plasma beams across a 30-2 kV voltage range, we examined how ion species and accelerating voltage influence block-face quality and ultrastructural fidelity. Curtaining imposed a species-dependent lower limit on accelerating voltage, with xenon and argon supporting uniform milling to 10 kV and oxygen and nitrogen to 15 kV. Monte Carlo simulations predicted substantially reduced ion penetration and atomic displacement under these species-specific low-kV conditions. Experimentally, lowering milling voltage suppressed charging and topographic artifacts, with the largest effects observed at heterogeneous interfaces. Oxygen and nitrogen produced greater membrane sharpness and organelle contrast than the noble gases, and these differences influenced machine-learning-based recognition of fine organelle features in 3D datasets. Together, these findings show that cryo-pFIB-SEM image quality cannot be optimized by accelerating voltage or ion species independently. Instead, milling performance reflects a tradeoff among surface fidelity, charging, contrast, and curtaining that depends on local specimen composition. Species-specific low-kV milling therefore provides a practical framework for matching cryo-pFIB-SEM acquisition conditions to the structural and biophysical properties of the biological target.