Cells, the basic structural and functional units, play an essential role in the development, aging, disease, and death of organisms. Since the first microscopic observation of cells by Robert Hooke in 1665, numerous advanced technical and theoretical methods have been developed over the past centuries to microscopically visualize cells, enabling a thorough analysis of life activities from the cellular to the molecular levels. Cells are composed of numerous macromolecular complexes with different sizes and diverse compositions. For example, the eukaryotic 80S ribosome is composed of large and small subunits, and each subunit contains various ribosomal RNA (rRNA) and ribosomal proteins. These complexes are usually considered as core signaling hubs to precisely control cellular structures and functions during various biological activities. Accordingly, cells can properly generate immediate responses to frequent environmental changes and distinct cellular stresses. Therefore, a mechanistic investigation of the structural assembly of these key signaling hubs and their functional regulation is necessary to improve our understanding of life activities and to identify potential therapeutic targets for disease treatment.

Currently, single-particle cryo-electron microscope (cryo-EM), which requires only a small number of samples for analysis and does not involve the use of crystals unlike traditional X-ray-based methods, is the most powerful tool in structural biology owing to its extremely high spatial resolution. However, precisely resolving a structure using cryo-EM involves purification or enrichment of the target biomolecules, which increases the risk of inconsistency between in vitro resolved structures and the native structures in cells. Notably, owing to the lack of molecular specificity, understanding the interactions among different molecules when resolving multi-component complexes is challenging. In addition, high-quality cryo-EM analyses depend on the computational averaging of thousands of images of identical particles with good homogeneity and are thus currently unsuitable for evaluating highly heterogeneous signaling hubs that determine cell fates.

Excitingly, super-resolution microscope (SRM) has emerged as an effective solution to these above-mentioned challenges. Fluorescence imaging is an indispensable technical tool for modern biological research owing to its molecular specificity, in situ visualization feature, and multiplex analysis ability. Super-resolution imaging, which overcomes the optical diffraction limit, is an efficient method for visualizing the arrangement and functions of biological hundred-nanometer signalosomes at the subcellular scale or even with single-molecule precision.

This review first introduces the basic principles and technical development of several major types of SRM, including stimulated emission depletion microscope (STED), structured illumination microscope (SIM), photoactivated localization microscope (PALM), stochastic optical reconstruction microscope (STORM), point accumulation for imaging in nanoscale topography (PAINT), DNA-PAINT, and minimal photon fluxes (MINFLUX). These tools have facilitated precise visualization of various biological activities and targets at remarkably high temporal and/or spatial resolutions, even reaching the molecular or angstrom scale in some extreme cases (Figs.1-2). More importantly, to date, several representative SRM-based applications in life science research have been demonstrated. Through rationale optimization of the key steps in STORM (including structure preservation, fluorescence labeling, signal acquisition, and image analysis), Xin Chen’s group from Xiamen University first visualized the ordered organization of necrosomes at the nanoscale and revealed their underlying mechanism to effectively initiate MLKL (a mixed lineage kinase domain-like protein)-dependent necroptosis and to precisely control the transition between apoptosis and necroptosis in cells stimulated by tumor necrosis factor (TNF). Maria Pia Cosma’s group from the Barcelona Institute of Science and Technology employed single-molecule localization microscope (SMLM) to investigate genome organization, especially the formation of chromatin ring structures. They proposed that the transcription-dependent negative superhelix primarily drives the master molecule cohesin to generate ring structures in vivo. The team led by Ana J. Garcia-Saez from the University of Tübingend quantitatively imaged Bax- and Bak-mediated pores in the mitochondrial outer membrane during intrinsic cell apoptosis; they observed the interplay of apoptosis and inflammation by controlling the dynamics of the mitochondrial content release. Ardem Patapoutian’s group from the Scripps Research utilized the iPALM and MINFLUX technologies to directly visualize the conformational stages of the mechanosensitive channel PIEZO1 in complex cellular environments (Fig.3). Finally, based on the practical experience gained from our group’s efforts, we summarized some important strategies, such as methods to minimize reconstruction artifacts, improve labeling efficiency, and strengthen quantitative analysis of super-resolved images, to obtain high-quality super-resolution images using SMLM (Fig.4).

With the groundbreaking innovation of SMLM in the past two decades, our understanding of structural organization and functional regulation in multiple types of cells undergoing various biological activities has improved. Although the classic genetic and biochemical experiments revealed the fundamental cellular mechanisms, cell imaging provides more precise and intuitive information on molecular interactions in situ. Therefore, the spatial resolution of SMLM can be further improved up to the molecular level to precisely depict an informative signaling network for a variety of critical biological processes. Thus, considering the continuous development of SMLM and other SRM technologies, we believe that in situ nanoscale functional organization of key signaling hubs will become one of the most promising research areas in cell biology in the near future. In addition, SMLM is expected to revolutionize research in science and technology and lead to outstanding discoveries in the next 5-10 years.