A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography.
We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.
Masich
Sergej
S
Department of Cell and Molecular Biology, Karolinska Institutet, Box 285, SE-17177 Stockholm, Sweden.
Ostberg
Tove
T
Norlén
Lars
L
Shupliakov
Oleg
O
Daneholt
Bertil
B
D020285:Cryoelectron Microscopy / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D015603:Keratinocytes / Q000166:cytology / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure; D015203:Reproducibility of Results; D012867:Skin / Q000166:cytology / Q000648:ultrastructure*; D014057:Tomography, X-Ray Computed / Q000295:instrumentation / Q000379:methods*