Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to existing ExM protocols is the equipping of different kinds of molecules, with different kinds of anchoring moieties, so they can all be pulled apart from each other by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables proteins and RNAs to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and reduces cost (by 2-10-fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this united ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructures, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues and may support the visualization of other kinds of biomolecular species as well. uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.
Background: Using primary airway epithelial cells (AEC) is essential to mimic more closely different types and stages of lung disease in humans while reducing or even replacing animal experiments. Access to lung tissue remains limited because these samples are generally obtained from patients who undergo lung transplantation for end-stage lung disease or thoracic surgery for (mostly) lung cancer. We investigated whether forceps or cryo biopsies are a viable alternative source of AEC compared to the conventional technique.
Iron (Fe) availability limits photosynthesis at a global scale where Fe-rich photosystem (PS) I abundance is drastically reduced in Fe-poor environments. We used single-particle cryo-electron microscopy to reveal a unique Fe starvation-dependent arrangement of light-harvesting chlorophyll (LHC) proteins where Fe starvation-induced TIDI1 is found in an additional tetramer of LHC proteins associated with PSI in and . These cosmopolitan green algae are resilient to poor Fe nutrition.
Expansion microscopy is a super-resolution technique in which physically enlarging the samples in an isotropic manner increases inter-molecular distances such that nano-scale structures can be resolved using light microscopy. This is particularly useful in neuroscience as many important structures are smaller than the diffraction limit. Since its invention in 2015, a variety of expansion microscopy protocols have been generated and applied to advance knowledge in many prominent organisms in neuroscience, including zebrafish, mice, , and .
Nuclear lipids play roles in regulatory processes such as signaling, transcriptional regulation, and DNA repair. In this report, we demonstrate that nuclear lipids may contribute to Ki-67-regulated chromosome integrity during mitosis. In COS-7 cells, nuclear lipids are enriched at the perichromosomal layer and excluded from intrachromosomal regions during early mitosis, but are then detected in intrachromosomal regions during late mitosis, as revealed by TT-ExM, an improved expansion microscopy technique that enables high-sensitivity, super-resolution imaging of proteins, lipids, and nuclear DNA.
Diverse tissues in vivo present varying degrees of confinement, constriction, and compression to migrating cells in both homeostasis and disease. The nucleus in particular is subjected to external forces by the physical environment during confined migration. While many systems have been developed to induce nuclear deformation and analyze resultant functional changes, much remains unclear about dynamic volume regulation in confinement due to limitations in time resolution and difficulty imaging in PDMS-based microfluidic chips.
