
Research Background
The cell is the basic unit of body structure and function composed of different organelles and the cytoplasmic matrix. The biological activities such as the characterization of its activity, functional expression, and morphological structure are illustrated by microscopies. With the development of optical technology, fluorescence super-resolution microscopies break through the diffraction limit of light and enable high-definition imaging of organelles included mitochondria, lysosomes, and ribosomes. It could provide scientific basis for revealing basic biomedical issues such as cell division, cell differentiation, cellular senescence, cellular apoptosis, and cellular communication. For example, tumor cells have the characteristics of vigorous growth and rapid proliferation, while have the risk of metastasis. The multi-drug resistance, toxicity and side effects lead to poor therapeutic effects during the process of anti-cancer drug discovery and development. With the innovation of super-resolution microscopy technologies, more subtle subcellular structure are realized to visualize tracking and position. It provides new insight for revealing the mechanism of material exchange and signal transduction under organelle interactions.
Recently, A review paper of "Super-resolution microscopy reveals new insights into organelle interactions" published by Prof. Chao Zuo of Nanjing University of Science and Technology in Advanced Imaging. Based on the principles of fluorescence super-resolution technologies, this review systematically summarizes the status quo and challenges of the research on the morphology, function and interaction effects of classical membrane-structured and membraneless organelles based on fluorescence super-resolution imaging technologies, and discusses the currently existing problems and development.
Fig.1 Application of fluorescence super-resolution technology based on biomedicine
1. Fluorescence super-resolution microscopy
Super-resolution methods can also be identified by three well-known methods, including single-molecule localization microscopy (SMLM), stimulated emission depletion (STED), and structured illumination (SIM). Everyone has its own unique characteristics, but they are able to surpass the diffraction limit of light and achieve higher-resolution imaging in common. SMLM represented by STORM uses the single-molecule localization algorithm to realize the accurate localization of random scintillation fluorophores. The temporal resolution is conducive to expanding the in-depth application of the technology due to tens of thousands of frames in the process of reconstruction. Fluorescence molecules of STED are excited by the lossy light, the results of which that the imaging resolution and point spread function (PSF) are improved. Meanwhile, photobleaching is rigorous problem for STED. For SIM, the sample is illuminated by structured light and high-resolution images are reconstructed by diffraction images at different angles. It performs particularly well for live cell and is suitable for long-term dynamic observation. MINFLUX is based on the doughnut beam as the excitation light and high-precision localization of fluorescent molecules are located by comparing the central point position with the unknown fluorophore position. Meanwhile, the research of super-resolution imaging technology based on deep learning has become a new insight for complex biomedical applications.
Fig. 2 Principle and applications of fluorescence super-resolution microscopies including STORM (A) , SIM(B), STED (C) and MINFLUX (D).
2. Organelle interactions effects under fluorescence super-resolution microscopies
Fluorescence super-resolution microscopies have the advantages of real-time visualization, real-time imaging capability and multi-color labeling capability, which provides novel insights for basic biomedicine such as organelle membrane junctions, interaction effects, cell response mechanisms, and so on. Remarkingly, they will pushes forward the high-speed development of modern biology. Organelles are micro-organs with certain morphology, structure and function. They maintain the dynamic physiological balance through complex interactions. The emergence of fluorescence super-resolution microscopy provides a new means for us to deeply study the interaction effects at the subcellular level. For membrane-structured organelles, membrane-membrane contact sites (MCS) plays a central role in carrying out material exchange and interaction effects. Mitochondria in live cells was tracked by SIM over a long period and the the key behaviors of mitochondria formation by fusion and division were visualized. The regulatory mechanism of lysosome combines with intracellular autophagy to form autophagy lysosome, which regulates the quantity, morphology and microenvironment of molecular substances.Researchers used SIM/STED to achieve visual tracking of biological phenomena such as calcium regulation between mitochondria-lysosomes, energy metabolism, key factors of mitochondrial fusion and dissociation, cellular autophagy-associated proteins monitoring, and drug-regulated cellular autophagy lysosomes. Compared with traditional optical microscopy, dSTORM achieves imaging analysis of its network and tubular structures, especially for MCS (spatial distance of 10-25 nm) between mitochondria and endoplasmic reticulum. Membraneless organelles are "liquid-liquid" phase separation aggregates formed by weak polyvalent interactions, which are composed of biological macromolecules such as proteins and RNA. The researchers used 3D-SIM to analyze the spatial fluorescence distribution of the transcriptional gene rDNA, and discovered the spatial distribution was inconsistent with that of rRNA. It further developed the spatial proteomics rapidly. Researchers achieved single-molecule activity and real-time dynamic localization of the ribosome (20-30 nm) substructure and its cell-matrix interaction effects under pathological conditions based on STORM. Moreover, subcellular organelle structures such as centrioles and centromeres were imaged by fluorescence super-resolution microscopies, assisting biologists to explore their importance in regulating cellular activity and organelle interaction effects.
Fig. 3 Analysis of interaction effects between membranous organelles by super-resolution microscopies. It included the interaction effect between mitochondria and lysosome (A), endoplasmic reticulum (B), nucleus (C), endoplasmic reticulum and Golgi (D).
Fig. 4 Analysis of interaction effects between membrane-free organelles by super-resolution imaging
Conclusion and Outlook
Compared with traditional fluorescence micrscopies, fluorescence super-resolution microscopy has the advantages of high resolution, high sensitivity, real-time imaging capability and multicolor labeling. This technique can not only obtain the morphology and spatial distribution of biomolecules, but also the interaction effects of different organelles during cellular dynamics and nanoparticle drug tracking importantly. The acquisition and reconstruction requires a large number of samples and time, the results of which limits the ability to analyze in real time. To overcome these challenges, researchers developed image processing methods based on deep learning. In addition, the combination of multimodal imaging maximizes the sensitivity and imaging quality of each super-resolution technique. It requires good biocompatibility of fluorescent probes during the process of live cell, which is another challenge for the development and applications of this technique. In conclusion, the performance of fluorescence super-resolution microscopies are comprehensively improved by algorithms, machine learning, device updates, and fluorescent probes. So they will promote advancing scientists' discovery of more delicate biomedical problems in cell biology and neuroscience to explore more mysteries of cellular life.