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Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) visualize either specific labels or anatomical ultrastructure but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical imaging method that combines ~16-24-fold linear expansion with fluorescent pan-stainings of proteins and lipids (providing EM-like ultrastructural context), and immunolabeling (for molecular imaging). We demonstrate the versatility of this approach by imaging synaptic and cell-specific antibodies in the ultrastructural 3D context of pre and postsynaptic densities, neuropil nanoarchitecture, and cellular organelles in dissociated neuron cultures and mouse brain tissue sections. Furthermore, we demonstrate tracing of neuronal circuitry from pan-ExM-t image volumes, suggesting that any laboratory with access to a confocal microscope can now localize specific molecules within nanoscale cellular and circuit contexts.

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus

The transition to parenthood brings significant changes in behavior toward offspring. For instance, in anticipation of their offspring, male mice shift from infanticidal to caregiving behaviors. While the release of oxytocin from the paraventricular hypothalamus (PVH) plays a critical role in paternal caregiving, it does not fully account for the entire behavioral shift. The specific downstream neurons and signaling mechanisms involved in this process remain obscure. Here, we demonstrate that PVH vasopressin neurons also essentially contribute to a paternal behavioral shift. This vasopressin signal is partially transmitted through oxytocin receptors (OTRs) expressed in the anterior commissure and medial nuclei of the preoptic area. These OTR-expressing neurons receive inputs from both PVH oxytocin and vasopressin neurons and are responsible for expressing paternal caregiving behaviors. Collectively, this non-canonical vasopressin-to-OTR crosstalk within specific limbic circuits acts as a pivotal regulator of paternal behavioral changes in mice.

Imaging Methods: CSV files pulsed illumination recorded image

Organisms: Mus musculus

Glioblastomas function as intricate cellular networks that extend into the surrounding brain tissue facilitating long distance communication. This malignant connectivity spans from the tumor core to remote infiltration zones, supporting the concept of glioblastoma as a whole-brain disease. With growing ethical concerns in biomedical research and the inherent limitations of animal models in recapitulating human glioblastoma biology, there is an increasing demand for human ex vivo platforms capable of capturing the full infiltration spectrum from the tumor core to single-cell dispersion. Here, we present a three-dimensional (3D), fully human ex vivo glioblastoma model (”Core2Edge”) that replicates this extensive infiltration range while preserving the intratumoral heterogeneity of the original tumor. This model involves implanting fluorescently labeled human glioblastoma organoids (GBOs) into organotypic human brain slices, maintaining the genetic integrity and cytoarchitecture of both brain and tumor. By combining tissue expansion with light-sheet fluorescence microscopy, we achieve super-resolution, 3D imaging of the entire GBO-brain slice model. This approach allows for the study of initial infiltration steps, in-depth analysis of the invasive front, exploration of cell-cell interactions between tumor cells and the tumor microenvironment, and offers a platform for drug screening and testing, reducing the need for animal models. Once GBOs are prepared, the protocol takes approximately 7-12 days. Key steps include brain slice preparation (~4-6 h, depending on quantity), one day for initial culture before GBO staining and transplantation, a variable culture period (up to 10 days), and fixation (~8 h). Additional time is required for downstream analyses, including imaging, sequencing, and proteomics.

Organisms: Homo sapiens

MEIOC prevents continued mitotic cycling and promotes meiotic entry during mouse oogenesis: In multicellular organisms, germ cells’ transformation into haploid gametes requires that they transition from mitosis to meiosis, whereby they stop mitotic cycling and enter the meiotic cell cycle. In mammals, transcriptional activator STRA8-MEIOSIN mediates the decision to enter the meiotic cell cycle by triggering the G1-to-meiotic S phase transition. However, the molecular basis by which mammalian germ cells prevent continued mitotic cycling before entering the meiotic cell cycle remains unclear. Here, we investigate MEIOC’s role in the mitosis-to-meiosis transition during mouse oogenesis by analyzing proliferation, cell cycle transcriptomics, and cell cycle-associated protein expression. MEIOC was previously shown to destabilize mRNA and repress the mitotic program after meiotic entry. Here, we demonstrate that MEIOC prevents continued mitotic cycling prior to meiotic entry in oogenic cells. We find that the mitosis-to-meiosis transition involves the repression of G1/S cyclin CCNA2 at the transcript and protein levels, and that MEIOC downregulates CCNA2 protein expression. In addition, MEIOC promotes entry into meiotic S phase by increasing Meiosin transcript abundance and consequently activating the STRA8-MEIOSIN transcription factor. Given that STRA8-MEIOSIN upregulates Meioc expression, MEIOC and STRA8-MEIOSIN form a positive feedback loop to reinforce timely meiotic initiation. We also demonstrate that BMP signaling halts mitotic cycling and promotes meiotic entry by upregulating MEIOC. We conclude that, in mouse oogenic cells, the transition from mitosis to meiosis occurs as two molecularly regulated steps– (i) halt of mitotic cycling and (ii) entry into the meiotic cell cycle – and that MEIOC modifies the cell cycle program to facilitate both steps in this transition. This study was published in: Ushuhuda EG*, Nguyen JT*, Pfaltzgraff NG, Wenner SM, Kofron M, Mikedis MM. MEIOC prevents continued mitotic cycling and promotes meiotic entry during mouse oogenesis. Development. 2025 Nov 25:dev205037. PMID: 41287933. * equal contribution

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus

Radiation damage remains a fundamental limitation in cryo-electron microscopy (cryo-EM), constraining the total electron dose that can be used and thus hindering high-resolution imaging of biological specimens. Recent studies have proposed that temporally structured or pulsed electron beams could reduce radiation damage by allowing time for energy dissipation between individual electron interactions. To evaluate this hypothesis, we conducted a systematic investigation using a radio-frequency (RF) driven 300 kV Titan Krios microscope equipped with cold field emission gun (c-FEG) to generate highly regular pulsed electron beams for specimens under cryogenic conditions. We compared radiation damage in three representative samples: paraffin 2D crystals, bacteriorhodopsin (purple membrane) 2D crystals, and plunge-frozen tobacco mosaic virus (TMV) in vitreous ice, under both pulsed and conventional random illumination, while keeping all other imaging conditions constant. Radiation damage was quantified by tracking the decay of computed diffraction intensities to determine the critical dose (Ne). We observed no statistically significant difference in critical dose between pulsed and random illumination across all 3 samples. Our findings provide a critical reference point for future development and evaluation of temporally modulated electron sources in cryo-EM instrumentation.

Imaging Methods: electron microscopy

Although it is well known that the morphology of Gram-negative rods changes on exposure to antibiotics, the morphology of antibiotic-resistant bacteria in the absence of antibiotics has not been widely investigated. Here, we studied the morphologies of 10 antibiotic-resistant strains of Escherichia coli and used bioinformatics tools to classify the resistant cells under light microscopy in the absence of antibiotics. The antibiotic-resistant strains showed differences in morphology from the sensitive parental strain, and the differences were most prominent in the quinolone-and β-lactam-resistant bacteria. A cluster analysis revealed increased proportions of fatter or shorter cells in the antibiotic-resistant strains. A correlation analysis of morphological features and gene expression suggested that genes related to energy metabolism and antibiotic resistance were highly correlated with the morphological characteristics of the resistant strains. Our newly proposed deep learning method for single-cell classification achieved a high level of performance in classifying quinolone-and β-lactam-resistant strains.

Imaging Methods: recorded image

Organisms: Escherichia coli

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell–cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, the authors analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, they showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Their results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

Imaging Methods: recorded image phase contrast microscopy time lapse microscopy fluorescence microscopy confocal microscopy

Organisms: Homo sapiens Canis lupus familiaris

Meiotic errors of relatively small chromosomes in oocytes result in egg aneuploidies that cause miscarriages and congenital diseases. Unlike somatic cells, which preferentially mis-segregate larger chromosomes, aged oocytes preferentially mis-segregate smaller chromosomes through unclear processes. Here, the authors provide a comprehensive three-dimensional chromosome identifying-and-tracking dataset throughout meiosis I in live mouse oocytes. This analysis reveals a prometaphase pathway that actively moves smaller chromosomes to the inner region of the metaphase plate. In the inner region, chromosomes are pulled by stronger bipolar microtubule forces, which facilitates premature chromosome separation, a major cause of segregation errors in aged oocytes. This study reveals a spatial pathway that facilitates aneuploidy of small chromosomes preferentially in aged eggs and implicates the role of the M phase in creating a chromosome size–based spatial arrangement.

Imaging Methods: recorded image confocal microscopy time lapse microscopy

Organisms: Mus musculus

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) was applied to measure EGFR receptor in resting and EGF-stimulated fixed HeLa cells. Pre-incubated antibody-nanobody construct was used to label EGFR. R3 docking strand was conjugated to the nanobody and complimentary imager strand was coupled to Cy3b fluorophore.

Organisms: Homo sapiens

Immunofluorescent images of male adolescent rat brain. Rats were exposed to 3,3'-dichloro-biphenyl-4-ol (sham, low, medium, and high groups) in a 28-day polymeric implant study. Four brain regions were analyzed.

Imaging Methods: confocal microscopy

Organisms: Rattus norvegicus

During ovariogenesis, more than two-thirds of germ cells are sacrificed to improve the quality of the remaining oocytes. However, the detailed mechanisms behind this selection process are not fully understood in mammals. Here, we developed a high-resolution, four-dimensional ovariogenesis imaging system to track the progression of oocyte fate determination in live mouse ovaries. Through this, we identified a cyst-independent oocyte phagocytosis mechanism that plays a key role in determining oocyte survival. We found that oocytes act as individual cells, rather than connected cyst structures, during ovarian reserve construction. In this process, dominant oocytes capture and absorb cell debris from sacrificed oocytes to enrich their cytoplasm and support their survival. Single-cell sequencing indicated that the sacrificed oocytes are regulated by autophagy. When oocyte sacrifice was inhibited using autophagy inhibitors, the pool of surviving oocytes expanded, but they failed to fully develop and contribute to fertility. Our study suggests that mammals have evolved a cyst-independent selection system to improve oocyte quality, which is essential for sustaining a long reproductive lifespan.

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus

Biomolecular condensates mediate dynamic compartmentalization of cellular processes. The multivalent interactions that underlie biomolecular condensation are often promoted by intrinsically disordered regions (IDRs) within proteins. While the role of IDRs in biomolecular condensates is well appreciated, predicting whether an IDR forms condensates in cells remains challenging. Here, we developed a machine learning model to accurately predict the condensation behavior of IDRs, analyzing 215 IDRs from fusion oncoproteins in HEK293T cells. We identified distinct sequence-derived physicochemical features associated with condensation. Leveraging these data, our model predicts that ~12% of the ~13,000 IDRs in the human proteome are likely to form cellular condensates, establishing a robust framework for proteome-wide analysis of IDR-mediated biomolecular condensation. Notably, proteins with condensate-forming IDRs are significantly enriched in RNA processing and splicing functions and are predominantly localized to membrane-less organelles (MLOs), highlighting a central role of IDR-mediated biomolecular condensation in cellular organization and RNA biology.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens