<p>Fine-scale transmission electron microscopy (TEM), focused ion beam scanning EM (FIB), and EM tomography have opened a new window on the synaptic organization of the normal, developing and pathologically altered brain in experimental animals. Progress in the human brain has been slower, due to technical challenges and the problem of tissue availability from donors that underwent epilepsy or tumor surgery. The present manuscript is in part an overview of the geometry of synaptic boutons in surgical biopsy samples taken from human temporal lobe neocortex (‘hTLN’). Here, the number, size, and shape of active zones (the equivalent of functional neurotransmitter release sites) and the three functionally defined pools of synaptic vesicles were quantified, with comparisons to the same parameters in experimental animals. High-resolution TEM tomography further allowed new insights concerning the readily releasable pool of synaptic vesicles, one of the key structural elements in synaptic transmission and plasticity. The quantitative 3D models of synaptic boutons provide the basis for numerical and/or Monte Carlo simulations of various signal cascades underlying synaptic transmission that at least in humans are still only partially accessible for experiment. In a second focus, we provide a step-by-step walk-through with illustrations of basic methodology for tissue preparation and analysis, for both TEM and FIB-SEM, including a thorough discussion of the main advantages and disadvantages of the several techniques and the particular challenge of working with human tissue.</p>