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Focused ion beam (FIB) technique together with other advanced microscopic techniques was applied to study the early microstructural changes leading to fatigue crack initiation in cyclically strained polycrystals (nickel, 316L steel). Dislocation structures of persistent slip bands (PSBs) and surrounding matrix were investigated in detail using electron channeling contrast imaging (ECCI) technique (concurrently in the FIB cross-section and on the specimen surface) and simultaneously with the surface relief topography using transmission electron microscopy (TEM) of thin surface foils prepared by in situ lift-out technique. True shape of extrusions and intrusions and the path of initiated fatigue cracks were assessed in three dimensions by FIB micro-tomography. The role of twin boundary in cyclic strain localization and fatigue crack initiation is discussed. Principal advantages and some limitations of FIB technique in …