Background and Objective: Introduction of focused ion beam (FIB) for transmission electron microscopy (TEM) preparation had enhanced the understanding of materials’ interaction at nanoscale. However, this technique generates localized heat that may possibly have some effect on organic/vital structures during preparation of biological tissues. Therefore, the aim of this study was to investigate the effect of milling with Cryogenic-FIB on imaging the ultra-morphological features of dentin-resin interface bonded in a tooth and compare the findings to a room-temperature FIB prepared specimens. Methods: After cylindrical dentin cavities (3 mm diameter × 1.5 mm depth) were prepared on the occlusal surfaces of extracted, non-carious human premolar teeth, they were restored with Filtek P90 (Silorane) restorative system (3M ESPE, USA). To investigate the ultra-morphological features of resin-dentin interface, the bonded specimens were divided into 2 groups based on the preparation technique; (1) FIB preparation at room-temperature (RT), and (2) FIB preparation with cryogenic cooling (Cy). Later, each group was examined under TEM. Results: The obtained sections in RT group showed blurred scattered needle-like crystals above the resin-impregnated dentin. However, the orientation of these crisscross needle-like crystals and the ultramorphological features of the underlying dentin were more vivid and distinct in Cy group. Conclusion: Within the limitation of this in-vitro study, it could be concluded that combining FIB with cryogenic cooling had preserved the biological organic features of dentin due to minimized beam damage. The presented cryogenic technique should be considered in future FIB/TEM studies involving biological substrates. This research was supported by King Abdulaziz University.