Project description:Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer's disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

| S-EPMC10965902 | biostudies-literature

FIB-Milled Quartz Nanopores in a Sealed Nanopipette.

Project description:We report the use of laser-pulled quartz nanopipettes as a new platform for microfabricated nanopores. A quartz nanopipette is prepared on a laser puller and sealed closed prior to focused-ion beam (FIB) milling. A quartz nanopore can then be FIB-milled into the side walls of the sealed pipette and used to analyze single nanoparticles. This method is fast, reproducible and creates nearly cylindrical nanopores in ultrathin quartz walls with controllable diameter down to 66 nm. Both pore size and wall thickness can be readily controlled in the FIB milling process by adjusting milling parameters and milling at different locations along the pipette walls. FIB-milled quartz nanopores combine the advantages of the pipette pores and silicon chip-based membrane pores into one device while avoiding many of the challenges of two popular nanopore devices. First, they can be used as a handheld probe device like a quartz pipette. Second, the use of an ultrathin quartz membrane gives them superior electric property enabling low noise recording at a higher bandwidth and a highly focused sensing zone located at a farther distance away from the highly restricted tip region. The inner and outer diameters of the resulting pore can be precisely measured using scanning electron microscopy (SEM). As an application, FIB-milled side nanopores are used to study translocation of polystyrene nanoparticles. In addition to studying the dependence of translocation time on the pore length, we demonstrate detection of nanoparticles in parallel nanopores of different lengths and use finite-element simulation to confirm the identity of the two resulting populations. Our results show that FIB-milled side nanopores are a useful platform for future analytical applications like studying nanoparticle translocation dynamics.

| S-EPMC6707750 | biostudies-literature

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