Micromachining tools and correlative approaches for cellular cryo-electron tomography.
A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.
Rigort
Alexander
A
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Bäuerlein
Felix J B
FJ
Leis
Andrew
A
Gruska
Manuela
M
Hoffmann
Christian
C
Laugks
Tim
T
Böhm
Ulrike
U
Eibauer
Matthias
M
Gnaegi
Helmut
H
Baumeister
Wolfgang
W
Plitzko
Jürgen M
JM
D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D004023:Dictyostelium / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods*; D005629:Frozen Sections / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D020102:Mycobacterium smegmatis / Q000648:ultrastructure; D011328:Prions / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure
D011328:Prions