Imaging of glycans on living cell membranes is crucial for elucidating their biological functions. Although fluorescent labeling of membrane glycans is commonly achieved by covalently attaching fluorophores to metabolically incorporated, tagged modified sugars, this approach is suboptimal for functional analysis, as it detects all incorporated sugar analogs indiscriminately, rendering it more suitable for comprehensive profiling. The fundamental challenge highlighted in 2018, namely, that “specific fluorescent labeling of glycans is not yet achievable,” remains unresolved. Overcoming these limitations is essential to enable functional glycan imaging. In parallel, recent studies have begun to employ a super-resolution microscopy technique known as Glyco-PAINT to examine lectin distribution on cell membranes [1]. Moreover, a novel approach has been developed to infer glycan-galectin-3 complex formation by monitoring reductions in the diffusion coefficient of glycans following galectin-3 addition to Halo-tagged membrane proteins expressing fully synthesized N-type glycans on the cell surface (Fig. 23.1) [2]. Furthermore, the report published in 2018 emphasized that highly precise synthetic methods are required to fluorescently label glycolipid glycans without compromising their functional integrity (Fig. 23.1). In recent years, a variety of glycolipid probes have been synthesized, expanding the available repertoire to approximately 40 distinct probes [3, 4]. Concurrently, refined force field parameters for simulating glycolipid glycans (ranging from coarse-grained to all-atom molecular dynamics calculations) have been proposed, and related studies are now emerging in the literature [5].