Alternating Angle Milling Suppresses Streaking Artifacts in FIB-SEM Imaging

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bioRxiv. 2025 Sep 26;. doi: 10.1101/2025.09.25.678572

Alternating Angle Milling Suppresses Streaking Artifacts in FIB-SEM Imaging FIB-SEM Technology

Shtengel G, Qiu W, Aaron J, Crowe AS, Polilov AA, Karkali K, Bleck CK, Hess HH

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Abstract

Focused ion beam scanning electron microscopy (FIB-SEM) (1–3) has been used in life sciences to produce large volumetric datasets with high resolution information on ultrastructure of biological organisms. 3D image acquisition is accomplished by serial removal of thin layers of material using focused ion beam (FIB) milling followed by scanning electron microscopy (SEM) imaging.One of the challenges in the standard FIB-SEM imaging protocol is that FIB milling results in characteristic artifacts, known as “streaks” or “curtains”. These streaks are caused by non-uniform material removal forming long straight trenches parallel to the FIB milling direction. These artifacts get worse along the milling direction and ultimately limit size of the SEM field of view.Various methods have been proposed to mitigate the streaks in acquired images. While these techniques often provide noticeable visual improvement, the underlying problem remains. The structural information in the “streaked” areas is lost due to non-uniform material removal during milling and cannot be fully recovered.We propose a simple modification allowing for a significant reduction of milling non-uniformities of streaks. We demonstrate the effectiveness of this approach on various samples.

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Alternating Angle Milling Suppresses Streaking Artifacts in FIB-SEM Imaging

Abstract

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