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Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, the authors engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, they disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. The authors applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. They achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.

Imaging Methods: recorded image spinning disk confocal microscopy single-beam laser-scanning confocal microscopy (LSCM) structured illumination microscopy (SIM) (Fbbi:,00000332) lattice SIM

Organisms: Homo sapiens Chlorocebus aethiops

Functionally mature neural circuits are shaped during postnatal development by eliminating redundant synapses formed during the perinatal period. In the cerebellum of neonatal rodents, each Purkinje cell (PC) receives synaptic inputs from multiple (more than 4) climbing fibers (CFs). During the first 3 postnatal weeks, synaptic inputs from a single CF become markedly larger and those from the other CFs are eliminated in each PC, leading to mono-innervation of each PC by a strong CF in adulthood. While molecules involved in the strengthening and elimination of CF synapses during postnatal development are being elucidated, much less is known about the molecular mechanisms underlying CF synapse formation during the early postnatal period. The authors show experimental evidence that suggests that a synapse organizer, PTPdelta, is required for early postnatal CF synapse formation and the subsequent establishment of CF to PC synaptic wiring. They showed that PTPdelta was localized at CF-PC synapses from postnatal day 0 (P0) irrespective of the expression of Aldolase C (Aldoc), a major marker of PC that distinguishes the cerebellar compartments. They found that the extension of a single strong CF along PC dendrites (CF translocation) was impaired in global PTPdelta knockout (KO) mice from P12 to P29-31 predominantly in PCs that did not express Aldoc [Aldoc (–) PCs].

Imaging Methods: recorded image confocal microscopy

Organisms: Mus musculus

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, the authors developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Imaging Methods: recorded image fluorescence microscopy confocal microscopy

Organisms: Homo sapiens

Periodic organ arrangements occur during growth and development and are widespread in both animals and plants. In bilaterian animals, repetitive organs are typically arranged periodically in a two-dimensional space defined by two body axes. In contrast, radially symmetrical animals and plants exhibit organ arrangements in three-dimensional space—around the body axis in animals and around the stem in plants. While the principles of periodic organ arrangement have been extensively studied in bilaterians, investigations in radially symmetrical animals remain limited. In the present study, the authors combined live imaging, quantitative analysis, and mathematical modeling to elucidate periodic organ arrangement in a radially symmetrical animal, Coryne uchidai (Cnidaria, Hydrozoa). The polyps of C. uchidai simultaneously formed multiple tentacles in a regularly spaced, ring-like pattern exhibiting radial symmetry. Multiple such rings appeared periodically along the body and largely maintained symmetrical organization. Additionally, the researchers observed polymorphisms in symmetry types—specifically tri-, tetra-, and pentaradial symmetries—occurring as individual variations. Notably, the type of radial symmetry was positively correlated with polyp diameter, with pentaradial polyps exhibiting a larger diameter than tetra- or triradial ones. Their mathematical model suggested that the selection of size-dependent radial symmetry is governed by activation-inhibition dynamics and positional information emanating from the mouth that guides tentacle initiation.

Imaging Methods: recorded image confocal microscopy time lapse microscopy

Organisms: Coryne uchidai

The authors established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of ×2 and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, they built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 µm across an FOV of approximately 1.5×1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. They applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. They also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hr, visualizing the movement of over 4.0×105 vascular endothelial cells and quantitatively analyzing their dynamics. Their results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

Imaging Methods: recorded image fluorescence microscopy AMATERAS-2w AMATERAS-1 spinning disk confocal microscopy AMATERAS-2c

Organisms: Homo sapiens Mus musculus Coturnix japonica

Reprocessed images from LINCS image dataset and Human Protein Atlas for training deep learning models.

Imaging Methods: fluorescence microscopy confocal microscopy

Organisms: Homo sapiens

Understanding the factors involved in chromatin dynamics is crucial for the study of biochemical processes in which distant genomic regions need to come in close proximity. Previous single locus tracking studies suggest that chromatin dynamics are linked to active transcription, but studies which compare the chromatin dynamics of different locations within a defined chromatin domain are still very limited. Here we used the ANCHOR3 DNA labeling system to track multiple cis-regulatory elements and non-regulatory control regions at different positions in the mouse Klf4 locus. We observe homogeneous chromatin motion of cis-regulatory elements and non-cis-regulatory control regions in Klf4 transcribing mESCs and their non-transcribing EpiLCs daughter cells. These observations challenge the notion that active transcription has a major effect on the locus dynamics of mammalian genes.

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus

Multiscale 3D image datasets of mammalian placental tissue

Imaging Methods: electron microscopy X-ray microscopy

Organisms: Mus musculus

Figure 7 Raw Data

Organisms: Setaria viridis

Eukaryotic chromosome segregation requires attachment of chromosomes to microtubules through the kinetochore so that chromosomes can align and move in mitosis. Kinetochores assemble on the centromere which is epigenetically defined by the histone H3 variant CENtromere Protein A (CENP-A). During DNA replication CENP-A is equally divided between replicated chromatids and new CENP-A nucleosomes are re-assembled during the subsequent G1 phase. How cells regulate the cell cycle timing of CENP-A assembly is a central question in the epigenetic maintenance of centromeres. CENP-A nucleosome assembly requires the Mis18 complex (Mis18α, Mis18β, and M18BP1) which is regulated in its localization to centromeres between metaphase and G1. Here, we define a new regulatory mechanism that works through phosphorylation of Xenopus laevis M18BP1 between metaphase and interphase. This switch disrupts binding of M18BP1 to CENP-A nucleosomes in metaphase, and when relieved enables M18BP1 binding to CENP-A nucleosomes in interphase. We show that this phosphorylation dependent mechanism regulates CENP-A nucleosome assembly. We propose that the phospho-regulated binding of M18BP1 to CENP-A nucleosomes restricts new CENP-A assembly to interphase.

Imaging Methods: spinning disk confocal microscopy

Organisms: Xenopus laevis

Original images

Imaging Methods: bright-field microscopy confocal microscopy

Organisms: Platynereis dumerilii

Figure 6 Raw Data

Organisms: Setaria viridis