Interferometric Image Scanning Microscopy for label-free imaging at 120 nm lateral resolution inside live cells

Light microscopy remains indispensable in life sciences for visualizing cellular structures and dynamics in live specimens. Yet, conventional fluorescence imaging can suffer from phototoxicity, limited labeling efficiency, or perturbation of biological function. Label-free techniques such as interferometric scattering microscopy (iSCAT) offer a powerful alternative by detecting nanoscale structures based on their light scattering, without the need for dyes or genetic tags. iSCAT has enabled high-sensitivity detection of single proteins and viruses on clean surfaces. More recently, its application to live cells has been extended by using confocal illumination and detection, allowing suppression of out-of-focus light, yielding subcellular structures with high contrast. This development laid the foundation for biologically relevant label-free imaging. Here, we introduce interferometric image scanning microscopy (iISM). This next-generation technique combines interferometric detection with image scanning microscopy to achieve about 120 nm lateral resolution while operating at tenfold lower incident illumination power per diffraction limited spot, significantly reducing photodamage while enhancing signal-to-noise and contrast. Using iISM, we are able to visualize intracellular organelles such as the endoplasmic reticulum, actin cytoskeleton, mitochondria, and vesicles in live cells at essentially unlimited observation times. Importantly, iISM can be readily combined with confocal fluorescence microscopy, enabling correlation of label-free dynamics and structural information with molecular specificity. Our approach opens new avenues for studying dynamic biological processes, such as host-pathogen interactions, intracellular trafficking, or cytoskeletal rearrangements, under label-free, near-native conditions. iISM thus offers a powerful new tool for high-resolution, low-impact imaging of live cells, paving the way for new biological insights.