Novel Hybrid Semiconductor-Cellular Standard for 3D FIB-SEM Nanotomography Analysis

Focused ion beam scanning electron microscopy (FIB-SEM) nanotomography is a powerful tool to achieve high-resolution 3D imaging. The reliability of the FIB-SEM acquisition is dependent on the instrument's stability. The ability to recognize disruptions in the acquired data is paramount to its use as a metrology tool. This investigation uses a commercial unbonded semiconductor die to evaluate the FIB-SEM performance across multiple ZEISS Crossbeam systems. By culturing adherent cells or drop-casting suspended cells onto the die, it can also be used to create a hybrid semiconductor-cellular standard that can evaluate FIB-SEM acquisitions containing biological material. The uniform tungsten plug array within the sublayers of the die's bond pad architecture allows for visualization of discontinuities within the acquisition. Abnormalities of the tungsten plug position and morphology correlate with deviations in the target slice thickness. The misalignment of the FIB-SEM dataset is quantified using an affine transform by comparing the xz reconstructed tungsten plug array to the reference plug arrangement. This misalignment can be utilized to appropriately resize the pixel data and may indicate the potential need for microscope recalibration. Overall, this workflow proposes a customizable standard sample that applies to multiple FIB-SEM systems, regardless of the manufacturer, to improve FIB-SEM nanotomography.