Qionghua Shen

Somatic oncogenic mutations are typically defined by their molecular alterations, yet how they reorganize cellular architecture within intact tissues remain largely unknown. Here, we demonstrate that distinct oncogenic drivers produce unique multiscale architectural phenotypes that can be quantitatively resolved in intact liver tissue. Using iterative expansion microscopy, multiscale light-sheet imaging, and three-dimensional morphometric analysis, we systematically mapped structural remodeling from single-cell morphology to mitochondrial architecture in mosaic mouse models of hepatocellular oncogene activation. NRAS and CTNNB1 induced fundamentally different morphological programs. NRAS activation drove extensive remodeling of cell morphology, membrane curvature, and surface irregularity, whereas CTNNB1 activation largely preserved global cell morphology while selectively altering mitochondrial organization and shape. We first established a zonation-aware reference state for interpreting oncogene-associated organelle remodeling by resolving mitochondrial differences between periportal and pericentral hepatocytes. Within this framework, CTNNB1 activation shifted mitochondrial features toward a pericentral-like state, consistent with the role of Wnt/β-catenin signaling in hepatic zonation and metabolic identity. Furthermore, integrating cellular morphology, membrane geometry, and mitochondrial architecture improved discrimination of oncogenic states beyond any individual structural feature, demonstrating that mutation-specific phenotypes arise through coordinated remodeling across multiple biological scales. Together, these findings establish multiscale structural phenotyping as a framework for linking oncogenic genotype to three-dimensional cellular organization and reveal that distinct oncogenic drivers remodel different architectural compartments during liver oncogene activation.

Altair-dvOPM is an open-access direct-view oblique plane microscope designed for large-field, three-dimensional imaging of cleared and expanded tissue sections. By combining photographic-lens-based detection, externally launched oblique illumination and precision-registered modular baseplates, the system achieves micrometer-scale lateral resolution over a ~5.4 mm field of view without custom objectives or highly specialized alignment procedures. We demonstrate imaging across scales, from subcellular structures in expanded cells to centimeter-scale expanded tissue sections, and provide documentation, CAD files, Zemax models and open-source control software to support replication and extension.

Despite advances in fluorescence microscopy, spectral overlap and limited resolution hinder the dense mapping of the cellular ultrastructure. To overcome these challenges, we developed Cy-ExM, a high-plex imaging strategy that integrates optimized cryo-fixation for antigen preservation, expansion microscopy, and iterative immunofluorescence labeling. Using oblique plane microscopy, we perform three-dimensional super-resolution imaging of 20 biological targets encompassing the full cellular volume of individual mammalian cells.

Metastatic glioblastoma multiforme (GBM) is known for its dismal prognosis due to the dissemination of single cells throughout the brain parenchyma and along white matter tracts, resulting in heightened resistance to therapies. Understanding the intricate relationship between cell migration, physical confinement, and chemotherapeutic resistance in GBM is imperative for advancing treatment strategies. In this study, we employed G55, a representative migratory GBM cell line, to investigate this phenomenon. We generated three distinct cell populations: (1) cells migrating without confinement, assessed via the Scratch assay; (2) cells migrating a short distance (10 μm) under confinement, examined through the Transwell assay; and (3) cells migrating long distances (>100 μm) under confinement, studied using the Microchannel assay. Comparative analyses of protein expression profiles and chemotherapy sensitivity among these groups revealed that migration combined with physical confinement plays a pivotal role in augmenting chemotherapeutic resistance in interstitial invasive cancer cells. Moreover, we demonstrate the utility of the microchannel device, which facilitates controlled cell migration under physical confinement, as an effective in vitro tool for investigating metastatic cancer and associated treatment resistance. This study sheds light on the mechanisms underlying GBM progression and highlights potential avenues for therapeutic intervention.

We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of Axially Swept Light-Sheet Microscopy (ASLM) that achieves a field of view, measuring 774 x 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of ∼420 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.

Ultrasound imaging and ultrasound‐mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need for microbubbles, which cannot transverse many biological barriers due to their large size. Here, the authors introduce 50‐nm gas‐filled protein nanostructures derived from genetically engineered gas vesicles(GVs) that are referred to as _{50 nm}GVs. These diamond‐shaped nanostructures have hydrodynamic diameters smaller than commercially available 50‐nm gold nanoparticles and are, to the authors’ knowledge, the smallest stable, free‐floating bubbles made to date. _{50 nm}GVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected _{50 nm}GVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen‐presenting cells adjacent to lymphocytes. The authors anticipate that _{50 nm}GVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas‐filled nanomaterials.

Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy. GV engineering has multiple applications, including reporter gene imaging, acoustic control and payload delivery. GVs often cluster into a honeycomb pattern to minimize occupancy of the cytosol. The underlying molecular mechanism and the influence on cellular physiology remain unknown. Using genetic, biochemical and imaging approaches, here we identify GvpU from Priestia megaterium as a protein that regulates GV clustering in vitro and upon expression in Escherichia coli. GvpU binds to the C-terminal tail of the core GV shell protein and undergoes a phase transition to form clusters in subsaturated solution. These properties of GvpU tune GV clustering and directly modulate bacterial fitness. GV variants can be designed with controllable sensitivity to GvpU-mediated clustering, enabling design of genetically tunable biosensors. Our findings elucidate the molecular mechanisms and functional roles of GV clustering, enabling its programmability for biomedical applications.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as _50nm GVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to our knowledge, the smallest stable, free-floating bubbles made to date. _50nm GVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected _50nm GVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. We anticipate that _50nm GVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.