In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.

Super-resolution microscopy has promoted the development of cell biology, but imaging proteins with low copy numbers in cellular structures remains challenging. The limited number of designated proteins within nuclear pore complexes (NPCs) impedes continuous observation in live cells, although they are often used as a standard for evaluating various SR methods. To address this issue, we tagged POM121 with Halo-SiR and imaged it using structured illumination microscopy with sparse deconvolution (Sparse-SIM). Remarkably, POM121-SiR exhibited more than six-fold fluorescence intensity and four-fold enhanced contrast compared to the same protein labeled with tandem-linked mCherry, while showing negligible photo-bleaching during SR imaging for 200 frames. Using this technique, we discovered various types of NPCs, including ring-like and cluster-like structures, and observed dynamic remodeling along with the sequential appearance of different Nup compositions. Overall, Halo-SiR with Sparse-SIM is a potent tool for extended SR imaging of dynamic structures of NPCs in live cells, and it may also help visualize proteins with limited numbers in general.

The development of super-resolution fluorescence microscopy is very essential for understanding the physical and biological fundamentals at nanometer scale. However, to date most super-resolution modalities require either complicated/costly purpose-built systems such as multiple-beam architectures or complex post-processing procedures with intrinsic artifacts. Achieving three-dimensional (3D) or multi-channel sub-diffraction microscopic imaging using a simple method remains a challenging and struggling task. Herein, we proposed 3D highly-nonlinear super-resolution microscopy using a single-beam excitation strategy, and the microscopy principle was modelled and studied based on the ultrahigh nonlinearity enabled by photon avalanches. According to the simulation, the point spread function of highly nonlinear microscopy is switchable among different modes and can shrink three-dimensionally to sub-diffraction scale at the photon avalanche mode. Experimentally, we demonstrated 3D optical nanoscopy assisted with huge optical nonlinearities in a simple laser scanning configuration, achieving a lateral resolution down to 58 nm (λ/14) and an axial resolution down to 185 nm (λ/5) with one single beam of low-power, continuous-wave, near-infrared laser. We further extended the photon avalanche effect to many other emitters to develop multi-color photon avalanching nanoprobes based on migrating photon avalanche mechanism, which enables us to implement single-beam dual-color sub-diffraction super-resolution microscopic imaging.

Limited by spatial and temporal resolution, traditional optical microscopy cannot image the delicate ultra-structure organelles and sub-organelles. The emergence of super-resolution microscopy makes it possible. In this review, we focus on mitochondria. We summarize the process of mitochondrial dynamics, the primary proteins that regulate mitochondrial morphology, the diseases related to mitochondrial dynamics. The purpose is to apply super-resolution microscopy developed during recent years to the mitochondrial research. By providing the right research tools, we will help to promote the application of this technique to the in-depth elucidation of the pathogenesis of diseases related to mitochondrial dynamics, assistdiagnosis and develop the therapeutic treatment.

Synaptic strength depends strongly on the subsynaptic organisation of presynaptic transmitter release and postsynaptic receptor densities, and their alterations are expected to underlie pathologies. Although synaptic dysfunctions are common pathogenic traits of Alzheimer\'s disease (AD), it remains unknown whether synaptic protein nano-organisation is altered in AD. Here, we systematically characterised the alterations in the subsynaptic organisation in cellular and mouse models of AD.
We used immunostaining and super-resolution stochastic optical reconstruction microscopy imaging to quantitatively examine the synaptic protein nano-organisation in both Aβ1-42-treated neuronal cultures and cortical sections from a mouse model of AD, APP23 mice.
We found that Aβ1-42-treatment of cultured hippocampal neurons decreased the synaptic retention of postsynaptic scaffolds and receptors and disrupted their nanoscale alignment to presynaptic transmitter release sites. In cortical sections, we found that while GluA1 receptors in wild-type mice were organised in subsynaptic nanoclusters with high local densities, receptors in APP23 mice distributed more homogeneously within synapses. This reorganisation, together with the reduced overall receptor density, led to reduced glutamatergic synaptic transmission. Meanwhile, the transsynaptic alignment between presynaptic release-guiding RIM1/2 and postsynaptic scaffolding protein PSD-95 was reduced in APP23 mice. Importantly, these reorganisations were progressive with age and were more pronounced in synapses in close vicinity of Aβ plaques with dense cores.
Our study revealed a spatiotemporal-specific reorganisation of synaptic nanostructures in AD and identifies dense-core amyloid plaques as the major local inductor in APP23 mice.

Fluorescence microscopy has become a routine tool in biology for interrogating life activities with minimal perturbation. While the resolution of fluorescence microscopy is in theory governed only by the diffraction of light, the resolution obtainable in practice is also constrained by the presence of optical aberrations. The past two decades have witnessed the advent of super-resolution microscopy that overcomes the diffraction barrier, enabling numerous biological investigations at the nanoscale. Adaptive optics, a technique borrowed from astronomical imaging, has been applied to correct for optical aberrations in essentially every microscopy modality, especially in super-resolution microscopy in the last decade, to restore optimal image quality and resolution. In this review, we briefly introduce the fundamental concepts of adaptive optics and the operating principles of the major super-resolution imaging techniques. We highlight some recent implementations and advances in adaptive optics for active and dynamic aberration correction in super-resolution microscopy.

Biological super-resolution microscopy is a new generation of imaging techniques that overcome the ~200 nm diffraction limit of conventional light microscopy in spatial resolution. By providing novel spatial or spatiotemporal information on biological processes at nanometer resolution with molecular specificity, it plays an increasingly important role in biomedical sciences. However, its technical constraints also require trade-offs to balance its spatial resolution, temporal resolution, and light exposure of samples. Recently, deep learning has achieved breakthrough performance in many image processing and computer vision tasks. It has also shown great promise in pushing the performance envelope of biological super-resolution microscopy. In this brief review, we survey recent advances in using deep learning to enhance the performance of biological super-resolution microscopy, focusing primarily on computational reconstruction of super-resolution images. Related key technical challenges are discussed. Despite the challenges, deep learning is expected to play an important role in the development of biological super-resolution microscopy. We conclude with an outlook into the future of this new research area.

Super-resolution microscopy (SRM) technology that breaks the diffraction limit has revolutionized the field of cell biology since its appearance, which enables researchers to visualize cellular structures with nanometric resolution, multiple colors and single-molecule sensitivity. With the flourishing development of hardware and the availability of novel fluorescent probes, the impact of SRM has already gone beyond cell biology and extended to nanomedicine, material science and nanotechnology, and remarkably boosted important breakthroughs in these fields. In this review, we will mainly highlight the power of SRM in modern biomedical science, discussing how these SRM techniques revolutionize the way we understand cell structures, biomaterials assembly and how assembled biomaterials interact with cellular organelles, and finally their promotion to the clinical pre-diagnosis. Moreover, we also provide an outlook on the current technical challenges and future improvement direction of SRM. We hope this review can provide useful information, inspire new ideas and propel the development both from the perspective of SRM techniques and from the perspective of SRM\'s applications.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.