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1. OsO ₂ as the Contrast‐Generating Chemical Species of Osmium‐Stained Biological Tissues in Electron Microscopy

   https://doi.org/10.1002/cbic.202400311  

   Li, Ruiyu; Wildenberg, Gregg; Boergens, Kevin; Yang, Yingjie; Weber, Kassandra; Rieger, Janek; Arcidiacono, Ashley; Klie, Robert; Kasthuri, Narayanan; King, Sarah_B (September 2024, ChemBioChem)

   Abstract Electron imaging of biological samples stained with heavy metals has enabled visualization of subcellular structures critical in chemical‐, structural‐, and neuro‐biology. In particular, osmium tetroxide (OsO₄) has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the process for contrast generation in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and therefore high‐resolution nanoscale imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with synaptic resolution, we have used PEEM to determine the nanoscale electronic structure of Os‐stained biological samples. Os(IV), in the form of OsO₂, generates nanoaggregates in lipid membranes, leading to a strong spatial variation in the electronic structure and electron density of states. OsO₂has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO₂in lipid membranes allows for strongly enhanced EM signals and conductivity of biological materials. The identification of the chemical species and understanding of the membrane contrast mechanism of Os‐stained biological specimens provides a new opportunity for the development of staining protocols for high‐resolution, high‐contrast EM imaging. 

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2. Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy

   https://doi.org/10.1073/pnas.2002200117  

   Heberle, Frederick A.; Doktorova, Milka; Scott, Haden L.; Skinkle, Allison D.; Waxham, M. Neal; Levental, Ilya (August 2020, Proceedings of the National Academy of Sciences)

   The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. 

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3. Polarization-dependent photoemission electron microscopy for domain imaging of inorganic and molecular materials

   https://doi.org/10.1063/5.0225765  

   Ghosh, Atreyie; Spellberg, Joseph L; King, Sarah B (September 2024, The Journal of Chemical Physics)

   Polarization-dependent photoemission electron microscopy (PD-PEEM) exploits spatial variation in the optical selection rules of materials to image domain formation and material organization on the nanoscale. In this Perspective, we discuss the mechanism of PD-PEEM that results in the observed image contrast in experiments and provide examples of a wide range of material domain structures that PD-PEEM has been able to elucidate, including molecular and polymer domains, local electronic structure and defect symmetry, (anti)ferroelectricity, and ferromagnetism. In the end, we discuss challenges and new directions that are possible with this tool for probing domain structure in materials, including investigating the formation of transient ordered states, multiferroics, and the influence of molecular and polymer order and disorder on excited state dynamics and charge transport. 

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4. Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy

   https://doi.org/10.1093/micmic/ozad077  

   Pérez-Garza, Janeth; Parrish-Mulliken, Emily; Deane, Zachary; Ostroff, Linnaea_E (August 2023, Microscopy and Microanalysis)

   Abstract Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction. 

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5. Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals

   https://doi.org/10.1364/BOE.411888  

   Samolis, Panagis_D; Langley, Daniel; O’Reilly, Breanna_M; Oo, Zay; Hilzenrat, Geva; Erramilli, Shyamsunder; Sgro, Allyson_E; McArthur, Sally; Sander, Michelle_Y (December 2020, Biomedical Optics Express)

   Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals). We present that the detectability of nano-sized cell membranes is enhanced to well below the optical diffraction limit since the membranes are found to act as thermal barriers. VIPPS offers a novel combination of chemical imaging and thermal diffusion characterization that paves the way towards label-free imaging of cell models and tissues as well as the study of intracellular heat dynamics. 

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