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Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo. A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

Probing the architecture, mechanism, and dynamics of genome folding is fundamental to our understanding of genome function in homeostasis and disease. Most chromosome conformation capture studies dissect the genome architecture with population‐ and time‐averaged snapshots and thus have limited capabilities to reveal 3D nuclear organization and dynamics at the single‐cell level. Here, we discuss emerging imaging techniques ranging from light microscopy to electron microscopy that enable investigation of genome folding and dynamics at high spatial and temporal resolution. Results from these studies complement genomic data, unveiling principles underlying the spatial arrangement of the genome and its potential functional links to diverse biological activities in the nucleus. Graphical Abstract Imaging technologies have been developed rapidly in recent years. This Review discusses emerging single‐cell imaging techniques and their applications in investigating genome folding and dynamics at high spatial and temporal resolution.

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. A kidney cell functions differently from a skin cell despite the fact that all the cells in one organism share the same DNA. This is because not all of the genes encoded within the DNA are active in the cells. Instead, cells can turn on just those genes that are specific to how that cell type works. One way that cells can regulate their genes is by using proteins called transcription factors that can bind to DNA to turn nearby genes on and off. When cells divide to form new cells, the DNA is condensed and gene activity is turned off. However, each dividing cell also has to ‘remember’ the program of genes that specifies its identity. After division, how do the cells know which genes to turn on and which ones to keep off? It was thought that the transcription factors attached to the DNA were all detached from it during cell division. Through studies in mouse embryonic stem cells, Teves et al. now show that this finding is largely an artifact of the methods used to study the process. In fact, many transcription factors still bind to and interact with DNA during cell division. This provides an efficient way for the newly formed cells to quickly reset to the pattern of gene activity appropriate for their cell type. Having found that many key transcription factors are still bound to DNA during cell division, the next challenge is to find out what role this binding plays in allowing cells to ‘remember’ their identity.

Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally.

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–fluorescence in situ hybridization (FISH), RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome. 3D ATAC-PALM integrates ATAC with super-resolution imaging for nanoscale views of the accessible genome. When combined with FISH, protein fluorescence and genetic perturbation, the method enables investigation of accessible chromatin in situ.

Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometers and up to several kilobases in resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtains comparable results of chromatin loops across datasets generated from diverse imaging technologies. SnapFISH is freely available at https://github.com/HuMingLab/SnapFISH . Multiplexed DNA FISH technologies are powerful tools to reveal chromatin spatial organisation. Here, the authors developed SnapFISH, a computational pipeline to identify chromatin loops from multiplexed DNA FISH data.

Mesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large-scale expansion while preserving therapeutic potency and maintaining small cell size. Conventional 2D culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution. We present an alternating 2D/3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations. Placenta-derived MSCs were cultured under optimized spheroid conditions, with extracellular matrix supplementation and chemically defined media to enhance viability. To address scalability, we developed RGD-functionalized alginate hydrogel tubes (AlgTubes) that enable dynamic transitions between adherent and spheroid states for continuous culture. Spheroid culture significantly reduced cell size and enhanced immunomodulatory function. The alternating 2D/3D protocol slowed MSC enlargement and senescence over multiple passages while preserving anti-inflammatory activity. Extracellular matrix supplementation and chemically defined media further improved cell viability. AlgTubes successfully supported the alternating culture strategy in a continuous and scalable format. The alternating 2D/3D culture system effectively overcomes limitations of conventional MSC expansion by mitigating enlargement, delaying senescence, and preserving both proliferative capacity and immunoregulatory potency. Combined with AlgTube technology, this work demonstrates a promising strategy for MSC manufacturing.

NUT carcinoma (NC) is an aggressive cancer with no effective treatment. About 70% of NUT carcinoma is associated with chromosome translocation events that lead to the formation of a BRD4::NUTM1 fusion gene. Because the BRD4::NUTM1 gene is unequivocally cytotoxic when ectopically expressed in cell lines, questions remain on whether the fusion gene can initiate NC. Here, we report the first genetically engineered mouse model for NUT carcinoma that recapitulates the human t(15;19) chromosome translocation in mice. We demonstrated that the mouse t(2;17) syntenic chromosome translocation, forming the Brd4::Nutm1 fusion gene, could induce aggressive carcinomas in mice. The tumors present histopathological and molecular features similar to human NC, with enrichment of undifferentiated cells. Similar to the reports of human NC incidence, Brd4::Nutm1 can induce NC from a broad range of tissues with a strong phenotypical variability. The consistent induction of poorly differentiated carcinoma demonstrated a strong reprogramming activity of BRD4::NUTM1. The new mouse model provided a critical preclinical model for NC that will lead to better understanding and therapy development for NC.

The rodent naive pluripotent state is believed to represent the preimplantation inner cell mass state of the developing blastocyst and can derive self-renewing pluripotent embryonic stem cells (ESCs) in vitro. Nevertheless, human ESCs exhibit epigenetic, metabolic, and transcriptomic characteristics more akin to primed pluripotent stem cells (PSCs) derived from the postimplantation epiblast. Understanding the genetic and epigenetic mechanisms that constrain human ESCs in the primed state is crucial for the human naive pluripotent state resetting and numerous applications in regenerative medicine. In this review, we begin by defining the naive and primed states in the murine model and compare the epigenetic characteristics of those states to the human PSCs. We also examine the various reprogramming schemes to derive the human naive pluripotent state. Finally, we discuss future perspectives of studying and deriving the human naive PSCs in the context of cellular engineering and regenerative medicine.