In this work, three novel TEM methodologies were used to characterize TSVs. First, we utilized the nano-beam diffraction technique to analyze the stress distribution within Si substrates. The keep-out zone can be determined in nano-scale. Second, the hollow-cone dark-field TEM technique was performed to enhance the image contrast of Cu grain features. Third, the TEM/EDS technique was utilized to examine the barrier layer continuity of through silicon TSVs. Both plan-view and cross-sectional view analysis were performed.