Position-controlled marker formation by helium ion microscope for aligning a TEM tomographic tilt series

► Formation of markers on a TEM specimen by using a helium ion microscope. ► Because of the position controllability, the markers could be placed efficiently. ► The method facilitates the identification of the markers in the alignment process. We formed nano-dots using a helium ion microscope (HIM) equipped with a gas injection system. Because of position controllability, the nano-dot markers could be placed efficiently on a specimen using the HIM. The sizes of the dots were controlled by changing the beam radiation time. We tried for the first time to form dots on a rod-shaped specimen to use them as markers for aligning a transmission electron microscope tomographic tilt series before reconstructing 3D images.