National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
P01NS092525
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
5P41GM103832
米国
National Institutes of Health/National Institute on Aging (NIH/NIA)
PO1AG054407
米国
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
F32NS086253
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
S10OD021600
米国
引用
ジャーナル: Structure / 年: 2020 タイトル: Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells. 著者: Gong-Her Wu / Patrick G Mitchell / Jesus G Galaz-Montoya / Corey W Hecksel / Emily M Sontag / Vimal Gangadharan / Jeffrey Marshman / David Mankus / Margaret E Bisher / Abigail K R Lytton-Jean ...著者: Gong-Her Wu / Patrick G Mitchell / Jesus G Galaz-Montoya / Corey W Hecksel / Emily M Sontag / Vimal Gangadharan / Jeffrey Marshman / David Mankus / Margaret E Bisher / Abigail K R Lytton-Jean / Judith Frydman / Kirk Czymmek / Wah Chiu / 要旨: Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging ...Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.