surgery-sample-processing-for-correlative-imaging-murine-pulmonary

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

Thermo Fisher Scientific

While fluorescence microscopy provides tools for highly specific labeling and sensitive detection, its resolution limit and lack of general contrast has hindered studies of cellular structure and protein localization. Recent advances in correlative light and electron microscopy (CLEM), including the fully integrated CLEM workflow instrument, the FEI CorrSight with MAPS, have allowed for a more reliable, reproducible, and quicker approach to correlate three-dimensional time-lapse confocal fluorescence data, with three-dimensional focused ion beam-scanning electron microscopy data. Here we demonstrate the entire integrated CLEM workflow using fluorescently tagged MCF7 breast cancer cells.

A fully integrated, three-dimensional fluorescence to electron microscopy correlative workflow.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.

The solid-electrolyte interphase (SEI), a layer formed on the electrode surface, is essential for electrochemical reactions in batteries and critically governs the battery stability. Active materials, especially those with extremely high energy density, such as silicon (Si), often inevitably undergo a large volume swing upon ion insertion and extraction, raising a critical question as to how the SEI interactively responds to and evolves with the material and consequently controls the cycling stability of the battery. Here, by integrating sensitive elemental tomography, an advanced algorithm and cryogenic scanning transmission electron microscopy, we unveil, in three dimensions, a correlated structural and chemical evolution of Si and SEI. Corroborated with a chemomechanical model, we demonstrate progressive electrolyte permeation and SEI growth along the percolation channel of the nanovoids due to vacancy injection and condensation during the delithiation process. Consequently, the Si-SEI spatial configuration evolves from the classic 'core-shell' structure in the first few cycles to a 'plum-pudding' structure following extended cycling, featuring the engulfing of Si domains by the SEI, which leads to the disruption of electron conduction pathways and formation of dead Si, contributing to capacity loss. The spatially coupled interactive evolution model of SEI and active materials, in principle, applies to a broad class of high-capacity electrode materials, leading to a critical insight for remedying the fading of high-capacity electrodes.

Cedric Bouchet-Marquis

Thermo Fisher Scientific

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve