Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high-frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher-order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super-resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed.

The spectrum and incidence of gene fusions in papillary thyroid carcinoma (PTC) can differ significantly depending on the age of onset, histological subtype or radiation exposure history. In sporadic pediatric PTC, RET/PTC1-3 and AGK-BRAF fusions are common genetic alterations. The role of RET/PTC as a prognostic marker in pediatric PTC is still under investigation. We recently showed that AGK-BRAF fusion is prevalent in young patients (mean 10 years) and associated with specific and aggressive pathological features such as multifocality and lung metastasis. In this pilot study, we report a unique patient harboring three different foci: the first was positive for AGK-BRAF fusion, the second was positive for just RET/PTC3 fusion and the third was negative for both rearrangements. To investigate whether AGK-BRAF and RET/PTC3 are associated with genomic instability and chromatin modifications, we performed quantitative fluorescence in situ hybridization (Q-FISH) of telomere repeats followed by 3D imaging analysis and 3D super-resolution Structured Illumination Microscopy (3D-SIM) to analyze the DNA structure from the foci. We demonstrated in this preliminary study that AGK-BRAF is likely associated with higher levels of telomere-related genomic instability and chromatin remodeling in comparison with RET/PTC3 foci. Our results suggest a progressive disruption in chromatin structure in AGK-BRAF-positive cells, which might explain a more aggressive disease outcome in patients harboring this rearrangement.

Paraspeckles are nuclear bodies built on an architectural long noncoding RNA, NEAT1, and a series of studies have revealed their molecular components, fine internal structures and cellular and physiological functions. Emerging lines of evidence suggest that paraspeckle formation is elicited by phase separation of associating RNA-binding proteins containing intrinsically disordered regions, which induce ordered arrangement of paraspeckle components along NEAT1. In this review, we will summarize the history of paraspeckle research over the last couple of decades, especially focusing on the function and structure of the nuclear bodies. We also discuss the future directions of research on long noncoding RNAs that form \'RNP milieux\', large and flexible phase-separated ribonucleoprotein complexes.

Single-molecule imaging (SMI) of proteins in operation has a history of intensive investigations over 20 years and is now widely used in various fields of biology and biotechnology. We review the recent advances in SMI of fluorescently-tagged proteins in structural biology, focusing on technical applicability of SMI to the measurements in living cells. Basic technologies and recent applications of SMI in structural biology are introduced. Distinct from other methods in structural biology, SMI directly observes single molecules and single-molecule events one-by-one, thus, explicitly analyzing the distribution of protein structures and the history of protein dynamics. It also allows one to detect single events of protein interaction. One unique feature of SMI is that it is applicable in complicated and heterogeneous environments, including living cells. The numbers, location, movements, interaction, oligomerization, and conformation of single-protein molecules have been determined using SMI in cellular systems.

Mitochondrial dynamics refers to the processes maintaining mitochondrial homeostasis, including mitochondrial fission, fusion, transport, biogenesis, and mitophagy. Mitochondrial dynamics is essential for maintaining the metabolic function of mitochondria as well as their regulatory roles in cell signaling. In this review, we summarize the recently developed imaging techniques for studying mitochondrial dynamics including: mitochondrial-targeted fluorescent proteins and dyes, live-cell imaging using photoactivation, photoswitching and cell fusion, mitochondrial transcription and replication imaging by in situ hybridization, and imaging mitochondrial dynamics by super-resolution microscopy. Moreover, we discuss examples of how to choose and combine proper fluorescent dyes and/or proteins.

Label-free optical nano-imaging of dendritic structures and intracellular granules in biological cells is demonstrated using a bright and homogeneous nanometric light source. The optical nanometric light source is excited using a focused electron beam. A zinc oxide (ZnO) luminescent thin film was fabricated by atomic layer deposition (ALD) to produce the nanoscale light source. The ZnO film formed by ALD emitted the bright, homogeneous light, unlike that deposited by another method. The dendritic structures of label-free macrophage receptor with collagenous structure-expressing CHO cells were clearly visualized below the diffraction limit. The inner fiber structure was observed with 120 nm spatial resolution. Because the bright homogeneous emission from the ZnO film suppresses the background noise, the signal-to-noise ratio (SNR) for the imaging results was greater than 10. The ALD method helps achieve an electron beam excitation assisted microscope with high spatial resolution and high SNR.