The methodology termed scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) has been used in this work to study the uptake of citrate stabilized gold nanoparticles (AuNPs, average particle sizes of 23.5±4.0 nm) into tissue samples upon in vitro exposure of the dissected gills of the Ruditapes philippinarum marine bivalve to the nanoparticle suspensions. The STEM-in-SEM methodology has been optimized for achieving optimum resolution at SEM low voltage operating conditions (20-30 kV). Based on scanning microscope assessments and resolution testing (SMART), resolutions well below 10 nm were properly achieved working at magnifications over 100 kx, with experimental sample thickness between 300 and 200 nm. These relative thick slices appear stable under the beam and help avoid NPs displacement during cutting. We show herein that both localizing of the internalized nanoparticles and imaging of ultrastructural disturbances in gill tissues are strongly accessible thanks to the improved resolution, even at sample thicknesses higher than those normally employed in standard TEM techniques at higher voltages. Ultrastructural imaging of Bio-Nano features in bioaccumulation experiments have been demonstrated in this study