This article presents a method for reconstructing three-dimensional (3D) tissue nanostructure using serial block-face scanning electron microscopy (SBFSEM). The technique involves automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope (SEM). Backscattering contrast is used to visualize heavy-metal staining of tissue prepared using techniques routine for transmission electron microscopy (TEM). Low-vacuum conditions (20–60 Pa H2O) prevent charging of the uncoated block face, allowing for high-resolution imaging. The resolution is sufficient to trace even the thinnest axons and identify small organelles such as synaptic vesicles. Stacks of several hundred sections, 50–70 nm thick, have been obtained with a lateral position jitter of typically under 10 nm. This method enables the acquisition of electron-microscope-level 3D datasets necessary for the complete reconstruction of neuronal circuits. The technique overcomes the limitations of traditional methods, such as the need for manual handling of sections and the inability to achieve high-resolution 3D data over large volumes. The study demonstrates the feasibility of this approach, showing that it can provide high-contrast images from uncoated block faces and enable the reconstruction of 3D ultrastructural data with sufficient resolution for local neural circuit reconstruction. The method also addresses challenges such as residual charging, image alignment, and sectioning precision, offering a more efficient and automated alternative to conventional serial sectioning techniques. The results highlight the potential of SBFSEM for advanced biological research, particularly in the study of neural connectivity and cellular structures.This article presents a method for reconstructing three-dimensional (3D) tissue nanostructure using serial block-face scanning electron microscopy (SBFSEM). The technique involves automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope (SEM). Backscattering contrast is used to visualize heavy-metal staining of tissue prepared using techniques routine for transmission electron microscopy (TEM). Low-vacuum conditions (20–60 Pa H2O) prevent charging of the uncoated block face, allowing for high-resolution imaging. The resolution is sufficient to trace even the thinnest axons and identify small organelles such as synaptic vesicles. Stacks of several hundred sections, 50–70 nm thick, have been obtained with a lateral position jitter of typically under 10 nm. This method enables the acquisition of electron-microscope-level 3D datasets necessary for the complete reconstruction of neuronal circuits. The technique overcomes the limitations of traditional methods, such as the need for manual handling of sections and the inability to achieve high-resolution 3D data over large volumes. The study demonstrates the feasibility of this approach, showing that it can provide high-contrast images from uncoated block faces and enable the reconstruction of 3D ultrastructural data with sufficient resolution for local neural circuit reconstruction. The method also addresses challenges such as residual charging, image alignment, and sectioning precision, offering a more efficient and automated alternative to conventional serial sectioning techniques. The results highlight the potential of SBFSEM for advanced biological research, particularly in the study of neural connectivity and cellular structures.