Upgrading interferometric scattering microscopy with ensemble statistical analysis

Lee, Minsu; Hong, Seok‐Cheol; Cho, Minhaeng

doi:10.1002/bkcs.12800

Cited by 1 publication

(1 citation statement)

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“…In recent decades, remarkable advances in label-free microscopy, particularly quantitative interference imaging, have emerged. − Among these advances, interferometric scattering (iSCAT) microscopy, an advanced progeny of interference reflection microscopy (IRM) specifically for nanoparticle characterization, has since evolved to encompass a wide range of applications in various research fields, including single biomolecule studies, chemical and biological sensing as well as cell biology. − ,,− In recent years, we have also played a role in expanding the range of applications of iSCAT-based imaging. These contributions include measuring the orientation of anisotropic nanoparticles, achieving vibration-free, long-range determination of the axial position of nanoparticles, measuring the radius of spherical nanoparticles in an all-optical manner, and enabling versatile live-cell imaging that is highly sensitive, label-free, and capable of both high-speed and long-term observation. − While there are several review articles overviewing the experimental achievements of IRM-based cellular imaging and delving into the technical details of iSCAT, − in this Account, our objective is to provide a review elucidating the transformative potential of iSCAT-based microscopy in advancing the understanding of intracellular trafficking through the provision of intricate insights.…”

Section: Introductionmentioning

confidence: 99%

Label-Free Interference Imaging of Intracellular Trafficking

Park,

Lee,

Hong

et al. 2024

Acc. Chem. Res.

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Conspectus Intracellular cargo trafficking is a highly regulated process responsible for transporting vital cellular components to their designated destinations. This intricate journey has been a central focus of cellular biology for many years. Early investigations leaned heavily on biochemical and genetic approaches, offering valuable insight into molecular mechanisms of cellular trafficking. However, while informative, these methods lack the capacity to capture the dynamic nature of intracellular trafficking. The advent of fluorescent protein tagging techniques transformed our ability to monitor the complete lifecycle of intracellular cargos, advancing our understanding. Yet, a central question remains: How do these cargos manage to navigate through traffic challenges, such as congestion, within the crowded cellular environment? Fluorescence-based imaging, though valuable, has inherent limitations when it comes to addressing the aforementioned question. It is prone to photobleaching, making long-term live-cell imaging challenging. Furthermore, they render unlabeled cellular constituents invisible, thereby missing critical environmental information. Notably, the unlabeled majority likely exerts a significant influence on the observed behavior of labeled molecules. These considerations underscore the necessity of developing complementary label-free imaging methods to overcome the limitations of fluorescence imaging or to integrate them synergistically. In this Account, we outline how label-free interference-based imaging has the potential to revolutionize the study of intracellular traffic by offering unprecedented levels of detail. We begin with a brief introduction to our previous findings in live-cell research enabled by interferometric scattering (iSCAT) microscopy, showcasing its aptitude and adeptness in elucidating intricate nanoscale intracellular structures. As we delved deeper into our exploration, we succeeded in the label-free visualization of the entire lifespan of nanoscale protein complexes known as nascent adhesions (NAs) and the dynamic events associated with adhesions within living cells. Our continuous efforts have led to the development of Dynamic Scattering-particle Localization Interference Microscopy (DySLIM), a generalized concept of cargo-localization iSCAT (CL-iSCAT). This label-free, high-speed imaging method, armed with iSCAT detection sensitivity, empowers us to capture quantitative and biophysical insights into cargo transport, providing a realistic view of the intricate nanoscale logistics occurring within living cells. Our in vivo studies demonstrate that intracellular cargos regularly contend with substantial traffic within the crowded cellular environment. Simultaneously, they employ inherent strategies for efficient cargo transport, such as collective migration and hitchhiking, to enhance overall transport ratesintriguingly paralleling the principle and practice of urban traffic management. We also highlight the synergistic benefits of combining DySLIM with chemical-selective ...

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