National Institutes of Health/National Institute on Aging (NIH/NIA)
RF1AG065341
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
P30AG072979
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
P01AG066597
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
U19AG062418
米国
David and Lucile Packard Foundation
2019-69645
米国
Burroughs Wellcome Fund
1022785
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
RM1GM136511
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
F30AG077756
米国
National Institutes of Health/National Cancer Institute (NIH/NCI)
F30CA261198
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM130302
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32GM132039
米国
引用
ジャーナル: Nat Commun / 年: 2024 タイトル: Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling. 著者: Benjamin C Creekmore / Kathryn Kixmoeller / Ben E Black / Edward B Lee / Yi-Wei Chang / 要旨: Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. ...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.
凍結剤: ETHANE-PROPANE / チャンバー内湿度: 90 % / チャンバー内温度: 288 K / 装置: LEICA EM GP / 詳細: back blot for 6s before plunging.
Cryo protectant
20% glycerol, 1M trehalose
切片作成
集束イオンビーム - 装置: OTHER / 集束イオンビーム - イオン: OTHER / 集束イオンビーム - 電圧: 30 / 集束イオンビーム - 電流: 0.05 / 集束イオンビーム - 時間: 7200 / 集束イオンビーム - 温度: 113 K / 集束イオンビーム - Initial thickness: 100000 / 集束イオンビーム - 最終 厚さ: 400 集束イオンビーム - 詳細: The value given for _em_focused_ion_beam.instrument is Tescan S8000X. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
Raw images were gain normalize in serialEM. Raw images were high-pass filtered at 1000 pixels then vertical line filtered with a 95% tolerance of direction.