<p>Plasmodesmata (PD) are nano-channels in plant cell walls that connect adjacent cells, facilitating the symplastic transport of micro- and macromolecules. In certain tissues, specialized transport requirements drive the formation of intercellular channels in diverse morphologies, compositions and functions, including sieve plate pores, plasmodesmo-pore units, lateral sieve area pore, and flexible gateways. These channels, derived from PD, are referred to as PD-derived intercellular gateways (PdIGs) in this review. Studies of PD and PdIGs are crucial for understanding how plants achieve cell-to-cell and long-distance transport of specific cargos under various physiological conditions. Multiple types of specialized microscopy are essential for studying these nano-channels, enabling elucidating their structures, compositions, distribution patterns, frequencies, and permeability. In particular, transmission electron microscopy (TEM), scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM), and structural illumination microscopy (SIM) offer advantages for such specialized analysis. With the aid of antibodies, fluorescent dyes, custom-built molecules, and image processing technologies, microscopy can also capture dynamic details of PD/PdIGs beyond morphology features. This review examines the application of these microscopy in studying specific aspects of PD/PdIGs, discusses the merits and limitations of these methods, and evaluate their suitability in PD studying the results obtained from these microscopy studies enhance our knowledge of PD/PdIGs and form the basis of the current paradigm of PD-based plant communication.</p>