kinds of microscopes is required, finder grids may be used to facilitate the process of correlation. Prior to use, the grids are glowdischarged to increase their hydrophilicity. In our lab, the grids are
glow-discharged for 60 s with 15 mA negative discharge at
1 Â 10
À1 bar using an Emitech K100Â glow-discharging device.
Gold fiducials are coated with bovine serum albumin (BSA) to
secure a homogenous dissemination of the beads. In a 1.5 mL
Eppendorf tube, 1 mL of gold nanoparticles (generally 10–20 nm
in diameter) is mixed with 5% BSA (two such tubes are usually
prepared). After that, they are spun down at 14,000 Â g for 30 min.
The pellets of the two tubes are resuspended in ~80 μL of the
supernatant. This solution is subsequently diluted (7–8 times)
with the bacterial sample prior to freezing. The remaining solution
of gold nanoparticles can be kept for many weeks at 4
C. If the
fiducial markers aggregate around the cells because of the inherent
characteristics of the bacteria (e.g., S-layer or extracellular matrix),
they can be laid down on the grids prior to freezing. To do so,
proceed as described above but dilute with PBS instead of the
bacterial sample. From this diluted solution, pipette 3 μL onto
each glow-discharged grid held by autoclosing forceps. Incubate
the drop for approximately 1 min and carefully back-blot the drop
on the other side of the grid, making sure the grid is not damaged.
Leave to dry for several minutes. Optional screening under a binocular loupe can ensure the overall integrity of the grid. The grids
are then glow-discharged again with the same parameters.
2.1.3 Plunge Freezing
of Bacterial Cells
Samples are blotted either manually or automatically in the blotting
chamber of the Vitrobot. Temperature is usually kept at 22
C with
100% humidity. The blotting conditions (e.g., humidity, blotting
force, and time) have to be identified and optimized for each
sample. Once plunge-frozen, samples can be kept in liquid nitrogen
dewars for long-term storage.
2.2 Eukaryotic Cells
The choice of cell type is a critical consideration for cryo-ET. For
successful cryo-ET imaging of peripheral cellular thin edges and
extensions, some types of mammalian cells are better suited and
reveal extensively larger imageable areas of cytoplasm. In our experience, U2OS, 3T3, and fibroblasts spread optimally on EM grids
to reveal peripheral thin edges that are electron transparent. Otherwise, additional thinning steps, such as FIB-milling (see below) may
be required to gain access to the thicker regions of the cell body and
nucleus.
Choice of grid support is critical. Standard copper EM grids are
toxic to cells; therefore, an inert grid material such as gold should
be used. For cryo-CLEM work, it is recommended that finder grids
with reference markers such as letters, symbols, and numbers be
used to easily locate target cells imaged between light microscope
(LM) and EM modalities. Here, we include all the steps for a cryoCLEM workflow.
86
Mohammed Kaplan et al.
glow-discharged for 60 s with 15 mA negative discharge at
1 Â 10
À1 bar using an Emitech K100Â glow-discharging device.
Gold fiducials are coated with bovine serum albumin (BSA) to
secure a homogenous dissemination of the beads. In a 1.5 mL
Eppendorf tube, 1 mL of gold nanoparticles (generally 10–20 nm
in diameter) is mixed with 5% BSA (two such tubes are usually
prepared). After that, they are spun down at 14,000 Â g for 30 min.
The pellets of the two tubes are resuspended in ~80 μL of the
supernatant. This solution is subsequently diluted (7–8 times)
with the bacterial sample prior to freezing. The remaining solution
of gold nanoparticles can be kept for many weeks at 4
C. If the
fiducial markers aggregate around the cells because of the inherent
characteristics of the bacteria (e.g., S-layer or extracellular matrix),
they can be laid down on the grids prior to freezing. To do so,
proceed as described above but dilute with PBS instead of the
bacterial sample. From this diluted solution, pipette 3 μL onto
each glow-discharged grid held by autoclosing forceps. Incubate
the drop for approximately 1 min and carefully back-blot the drop
on the other side of the grid, making sure the grid is not damaged.
Leave to dry for several minutes. Optional screening under a binocular loupe can ensure the overall integrity of the grid. The grids
are then glow-discharged again with the same parameters.
2.1.3 Plunge Freezing
of Bacterial Cells
Samples are blotted either manually or automatically in the blotting
chamber of the Vitrobot. Temperature is usually kept at 22
C with
100% humidity. The blotting conditions (e.g., humidity, blotting
force, and time) have to be identified and optimized for each
sample. Once plunge-frozen, samples can be kept in liquid nitrogen
dewars for long-term storage.
2.2 Eukaryotic Cells
The choice of cell type is a critical consideration for cryo-ET. For
successful cryo-ET imaging of peripheral cellular thin edges and
extensions, some types of mammalian cells are better suited and
reveal extensively larger imageable areas of cytoplasm. In our experience, U2OS, 3T3, and fibroblasts spread optimally on EM grids
to reveal peripheral thin edges that are electron transparent. Otherwise, additional thinning steps, such as FIB-milling (see below) may
be required to gain access to the thicker regions of the cell body and
nucleus.
Choice of grid support is critical. Standard copper EM grids are
toxic to cells; therefore, an inert grid material such as gold should
be used. For cryo-CLEM work, it is recommended that finder grids
with reference markers such as letters, symbols, and numbers be
used to easily locate target cells imaged between light microscope
(LM) and EM modalities. Here, we include all the steps for a cryoCLEM workflow.
86
Mohammed Kaplan et al.