Fluorogenic Rhodamine Probes Enable High‐Resolution Visualization of Plasma Membrane Nanostructures

Abstract The plasma membrane exhibits diverse substructures, such as pseudopodia, membrane nanotubes, and migrasomes, that are essential for cellular communication and cargo transport. Imaging these fine structures remains challenging due to their nanoscale dimensions and limitations of existing fluorescent probes. Here, we report the development of two rhodamine‐based probes, RSD1 and RSD2 , incorporating anionic membrane‐anchoring groups and pyrrolidine auxochromes to enable wash‐free, serum‐compatible, long‐term plasma membrane imaging. RSD2 , in particular, demonstrates superior fluorogenicity, brightness, and photoswitching properties, facilitating high‐resolution imaging in both live and fixed cells. It selectively labels membrane substructures across diverse cell types and maintains membrane specificity in the presence of serum. RSD2 is compatible with advanced microscopy techniques including confocal microscopy, instant structured illumination microscopy (iSIM), and direct stochastic optical reconstruction microscopy (dSTORM), achieving up to 40 nm resolution. Using two‐color dSTORM, we visualize silica nanoparticle trafficking via membrane nanotubes and gondola‐like bulges in neuronal cells, marking the first such observation. RSD2 also enables imaging of migrasomes and retraction fibers, revealing dynamic membrane‐mediated transport processes. This probe offers a robust and versatile platform for investigating membrane architecture and function, with broad applicability in cell biology, nanomedicine, and super‐resolution imaging.