A sub-0.25 mu m defect characterization study was conducted by using in-line inspection machines to locate defects and focused ion beam system (FIB) equipped with a navigation tool(1-3) to generate cross-sectional transmission electron microscopy (TEM) samples(4-8). Two failure analysis cases regarding to invisible defects in optical microscope were reported in this work. One described the micro-trench formed at the bird's beak of field oxide, and the other one illustrated the etching pit formation during Poly-Si etching process.