Project description:To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ?300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.
Project description:In this work, the application of a time resolved multi-contrast beam tracking technique to the investigation of the melting and solidification process in metals is presented. The use of such a technique allows retrieval of three contrast channels, transmission, refraction and dark-field, with millisecond time resolution. We investigated different melting conditions to characterize, at a proof-of-concept level, the features visible in each of the contrast channels. We found that the phase contrast channel provides a superior visibility of the density variations, allowing the liquid metal pool to be clearly distinguished. Refraction and dark-field were found to highlight surface roughness formed during solidification. This work demonstrates that the availability of the additional contrast channels provided by multi-contrast X-ray imaging delivers additional information, also when imaging high atomic number specimens with a significant absorption.
Project description:Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.
Project description:Mutually exclusive cell fate determination of CD4 helper or CD8 killer T cells occurs in the thymus. These T-cell subsets are not believed to redirect other lineages. Here we showed that retinoic acid and transforming growth factor-?1 promoted the differentiation of CD8?? T cells from CD4 T cells in a Runx3-dependent manner. These cells were inferred to belong to immunoregulatory populations because subpopulations of CD8??+TCR?? T cells are known to suppress activated T cells, and mice with Runx3(-/-) T cells showed defects during recovery from experimental allergic encephalomyelitis. Our results demonstrate that CD4 T cells play fundamental roles in controlling immune reactions through promotion and attenuation. We accordingly anticipate that clarifying the mechanisms underlying this process will provide insights leading to autoimmune and immunodeficiency disease therapies.
Project description:Ferroptosis is an emerging cancer suppression strategy. However, how to select cancer patients for treating with ferroptosis inducers remains challenging. Here, we develop photochemical activation of membrane lipid peroxidation (PALP), which uses targeted lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation for reporting ferroptosis sensitivity in cells and tissues. PALP captured by BODIPY-C11 can be suppressed by lipophilic antioxidants and iron chelation, and is dependent on PUFA-lipid levels. Moreover, we develop PALPv2, for studying lipid peroxidation on selected membranes along the z axis in live cells using two-photon microscopes. Using PALPv1, we detect PUFA-lipids in multiple tissues, and validate a PUFA-phospholipid reduction during muscle aging as previously reported. Patterns of PALPv1 signals across multiple cancer cell types in vitro and in vivo are concordant with their ferroptosis susceptibility and PUFA-phospholipid levels. We envision that PALP will enable rapid stratification of ferroptosis sensitivity in cancer patients and facilitate PUFA-lipid research.
Project description:We have discovered that hard, electrical conductors (e.g., metals or graphite) can be adhered to soft, aqueous materials (e.g., hydrogels, fruit, or animal tissue) without the use of an adhesive. The adhesion is induced by a low DC electric field. As an example, when 5 V DC is applied to graphite slabs spanning a tall cylindrical gel of acrylamide (AAm), a strong adhesion develops between the anode (+) and the gel in about 3 min. This adhesion endures after the field is removed, and we term it as hard-soft electroadhesion or EA[HS]. Depending on the material, adhesion occurs at the anode (+), cathode (-), or both electrodes. In many cases, EA[HS] can be reversed by reapplying the field with reversed polarity. Adhesion via EA[HS] to AAm gels follows the electrochemical series: e.g., it occurs with copper, lead, and tin but not nickel, iron, or zinc. We show that EA[HS] arises via electrochemical reactions that generate chemical bonds between the electrode and the polymers in the gel. EA[HS] can create new hybrid materials, thus enabling applications in robotics, energy storage, and biomedical implants. Interestingly, EA[HS] can even be achieved underwater, where typical adhesives cannot be used.
Project description:The mechanical micro-environment influences cellular responses such as migration, proliferation, differentiation and apoptosis. Cells are subjected to mechanical stretching in vivo, e.g., epithelial cells during embryogenesis. Current methodologies do not allow high-resolution in situ observation of cells and tissues under applied strain, which may reveal intracellular dynamics and the origin of cell mechanosensitivity. A novel polydimethylsiloxane substrate was developed, capable of applying tensile and compressive strain (up to 45%) to cells and tissues while allowing in situ observation with high-resolution optics. The strain field of the substrate was characterized experimentally using digital image correlation, and the deformation was modeled by the finite element method, using a Mooney-Rivlin hyperelastic constitutive relation. The substrate strain was found to be uniform for >95% of the substrate area. As a demonstration of the system, mechanical strain was applied to single fibroblasts transfected with GFP-actin and whole transgenic Drosophila embryos expressing GFP in all neurons during live imaging. Three observations of biological responses due to applied strain are reported: (1) dynamic rotation of intact actin stress fibers in fibroblasts; (2) lamellipodia activity and actin polymerization in fibroblasts; (3) active axonal contraction in Drosophila embryo motor neurons. The novel platform may serve as an important tool in studying the mechanoresponse of cells and tissues, including whole embryos.
Project description:In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.
Project description:Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-β plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.
Project description:Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.