Until now, scientists have relied on fluorescence microscopy techniques to study intracellular cargo transport, a vital process to maintaining essential cellular functions. However, the effects of photobleaching and the visual isolation of cellular features from their environment limit the modality’s ability to glean information about the process and how it is conducted within the crowded cellular environment. Researchers at the Institute for Basic Science Center for Molecular Spectroscopy and Dynamics (IBS CMSD), in collaboration with Korea University, developed a label-free, cargo-tracing microscopy technique to address these challenges. The Cargo-Localization Interferometric Scattering (CL-iSCAT) microscope enables label-free, real-time observation of cargo trafficking in the submicron cellular environment and allows researchers to selectively monitor the dynamic movement of active cargos within living cells. Schematic representation of a cellular traffic pattern drawn with the road network in and around Seoul. Courtesy of the Institute for Basic Science. Instead of pursuing chemical contrast using fluorophores, CL-iSCAT uses the dynamic feature of transported cargos to differentiate the cargos from other cytoplasmic objects. It uses time-differential image analysis to precisely track the movements of hundreds of cargos simultaneously over an extended time. It collects data by detecting scattering signals from all types of cargos that are moving directionally along a cytoskeletal track over time. To identify cellular vesicles and learn how they appear in the iSCAT microscopy system, the researchers fed cells with 20-nm fluorescent polystyrene beads and visualized them with fluorescence and iSCAT microscopies simultaneously. The label-free, iSCAT cargo-tracing method revealed not only the dynamics of cargo transportation, but also the fine architecture of the actively used cytoskeletal highways and the long-term evolution of the associated traffic. The researchers acquired a large amount of localization data from all cargo positions during the approximately 30-min observation time. This enabled the team to reconstruct the architecture of cytoskeletal meshwork and visualize the temporal evolution in intracellular traffic in high spatial resolution. CL-iSCAT features a dual-modality system that integrates fluorescence imaging with iSCAT microscopy, allowing separate observation of specifically labeled cargos or subcellular structures against the unmarked cargos moving along the microtubular networks. CL-iSCAT provides images of label-free cargos with a resolution down to 15 nm. “Through the achievement of observing live cells at an ultrahigh resolution independent of fluorescence, we have established a novel paradigm for elucidating the intricate details of biological processes,” said professor Minhaeng Cho, who directs CMSD and co-led the work on CL-iSCAT. Visualization of intracellular cargo traffic using CL-iSCAT microscopy. Each image depicts the evolution of cargo transport in the lamellipodium region of a cell, tracked at 50 Hz for 180 seconds (equivalent to 9000 frames). The color legend in the cargo traffic map represents the number of mobile cargos counted at each pixel over 180 seconds, providing quantitative insights into traffic density on the cellular cytoskeleton. The cell boundary is indicated by the yellow dashed line. Courtesy of the Institute for Basic Science. The researchers observed that cells intrinsically have efficient transport strategies for avoiding an intracellular “traffic jam” by forming a train of cargos or collectively moving in the same direction. Over a short time-scale, the cargo traffic appeared to be entangled and stagnant, the researchers observed; but over a long time-scale, the cargos moved as a collective cluster. In some cases, two or more cargos were directly connected and moved together. The experiments strongly supported the hypothesis of intracellular transport by hitchhiking as one strategy that cells use to increase the overall transport rate of the cargos. The researchers further observed that cellular traffic mirrors the real-life roadway traffic encountered in daily life. Like people on their way to work during rush hour, cells experience jams and obstacles that block the progress of the cargo, including possible head-on collisions with other cargos and abrupt stops at the intersections of cytoskeletal highways. “It is particularly fascinating to discover several typical traffic events experienced by city commuters in the highly complex cellular world, but at micrometer scales,” said Cho. “In the future, we aim to delve deeper into the efficient transport strategies adopted by cells to overcome these challenges in transportation and their relevance to cellular phenomena.” A) Traffic jams at the intersection of the cytoskeleton in a local area within a cell. This figure captures a traffic congestion event evidenced by cargo crowding and suppressed mobility. (Left) Static background removed (SBR-)iSCAT. (Right) Time-differential (TD-)iSCAT imaging techniques. Notably, both bright and dark spots in the SBR-iSCAT image (highlighted by numbered yellow circles) correspond to dynamic cargos observed in the TD-iSCAT image. Within the red circle in the TD-iSCAT image, a cluster of cargos experiences a bottleneck. B) A total of 83 trajectories of cargo continuously tracked over 500 frames in 9000 consecutive SBR-iSCAT images are drawn. Among these trajectories, those of 49 cargos displaying subdiffusive motion are depicted in gray. Courtesy of the Institute for Basic Science. The CL-iSCAT microscope could be used to investigate viral infections and monitor the effects of anti-viral vaccines and drugs in real time. Since the size of typical viruses is a few tens of nanometers, it would be possible to use CL-iSCAT to visualize the entire viral process from the onset of infection to cell death. The high-speed imaging and enormous amount of cargo localization data provided by iSCAT will enable researchers to acquire a realistic picture of nanoscale logistics within living cells and investigate the evolution over time of nanoscopic cellular constituents at high spatiotemporal resolution. The integration of two complementary techniques — fluorescent microscopy and interferometric scattering microscopy — could help drive innovative research in cell biology, deepening scientific understanding of biological phenomena that occur within cells. “The development of an imaging technology enabling high-resolution and rapid observation of biological processes allows for an in-depth understanding of life from a molecular dynamics perspective,” professor Seok-Cheol Hong, who co-led the research, said. “Our new approach of long-term visualization holds great potential for groundbreaking medical discovery.” The research was published in Nature Communications (www.doi.org/10.1038/s41467-023-42347-7).