Using an electron microscope's scanning transmission mode (STEM) for collection of tomographic datasets is advantageous compared to bright field transmission electron microscopic (TEM). For image formation, inelastic scattering does not cause chromatic aberration, since in STEM mode no image forming lenses are used after the beam has passed the sample, in contrast to regular TEM. Therefore, thicker samples can be imaged. It has been experimentally demonstrated that STEM is superior to TEM and energy filtered TEM for tomography of samples as thick as 1 μm. Even when using the best electron microscope, adequate sample preparation is the key for interpretable results. We adapted protocols for high-pressure freezing of cultivated cells from a physiological state. In this chapter, we describe optimized high-pressure freezing and freeze substitution protocols for STEM tomography in order to obtain high membrane contrast.