Lignin-based binders have been explored and studied as a sustainable alternative to conventional petroleum-based materials. They are recognised for their potential environmental advantages, such as reducing the carbon footprint, promoting the valorisation of renewable resources, and, in some cases, even providing antioxidant properties. Nevertheless, their microstructural features and appearance, unlike those of virgin or polymer-based bitumen, remain largely unexplored and poorly understood, although microscopic techniques provide a powerful means to “reveal the invisible” features that govern binder behaviour, including potential binder indicator and morphological changes induced by additives or ageing. The objective of this study is to provide a method for integrating multi-scale microscopic evidence and compare bio-based binders vs bitumen, under controlled ageing procedures using complementary microscopic techniques (i.e., optical, CLSM, fluorescence) and coupled with FTIR for chemical interactions insights. The observations revealed distinct microstructural features across binders and ageing stages. Variations in microstructural aspects and the size and shape of bee structures reflected the influence of lignin type and ageing procedure, providing new insights into the lignin-bitumen interactions.