measuring a set density of cells in suspension using a cell
counter. Or the trypsinized cells in the 10 mL Falcon tube
can simply be pipetted onto the medium above the grids and
left to settle into the grid squares. A light microscope can be
used to evaluate if a suitable density has been achieved.
4. U2OS cells are grown on the grids for 15–20 h before plungefreezing.
2.2.4 Freezing Grids,
Including Adding 20 nm
Gold Beads
and Fluorescent
Microspheres
for Cryo-CLEM
1. The EM grids are taken out of the media with Vitrobot tweezers, which are placed into the Vitrobot (see Note 5). Blot the
excess media from the grid before adding 3 μL of 20 nm gold
solution and 0.5 μm TetraSpeck Microspheres (see Note 6).
2. We recommend manual blotting from the gold side of the grid
with all Vitrobot parameters set to 0. It is recommended that
the humidity of the Vitrobot chamber be at 95% when the grid
is blotted (see Notes 7 and 8).
3. After plunge-freezing, the grids are stored within grid boxes
cooled in clean liquid nitrogen.
4. Before cryo-LM or cryo-EM imaging, EM grids are clipped
into FEI autogrids.
2.2.5 Cryo-LM Imaging
for Cryo-CLEM Using
the FEI Cryostage
1. In our setup, an FEI cryostage modified to hold Titan Krios
cartridges is used, and liquid nitrogen is continuously pumped
into the stage by a Norton cryo-pump to maintain the temperature at 80 K. Wait approximately 40 min for the stage to
equilibrate and maintain its cryogenic temperature.
2. Load one grid into the stage at a time.
3. Collect a montage or a series of individual phase contrast and
fluorescent Z-stack images of mammalian cells growing flat
within the grid square and near the center of the grid
(Fig. 1). If your target protein is tagged with a green fluorescent protein (GFP) (see Note 9), collection of red, blue, and
green fluorescent images is recommended (see Note 10).
4. Evaluation of ice thickness should be made when imaging
mammalian cells using cryo-LM. Ice cracks and thick ice
around the circumference of the square are indications that
the ice is too thick around the cell. In addition, obvious cracks
in the carbon support close to target cells are not ideal and will
affect the stability of the cell when being tilted, which can lead
to suboptimal tilt-series alignment.
5. Align the fluorescent images to the fluorescent microspheres
observed in the phase contrast image (see Note 11).
88
Mohammed Kaplan et al.
counter. Or the trypsinized cells in the 10 mL Falcon tube
can simply be pipetted onto the medium above the grids and
left to settle into the grid squares. A light microscope can be
used to evaluate if a suitable density has been achieved.
4. U2OS cells are grown on the grids for 15–20 h before plungefreezing.
2.2.4 Freezing Grids,
Including Adding 20 nm
Gold Beads
and Fluorescent
Microspheres
for Cryo-CLEM
1. The EM grids are taken out of the media with Vitrobot tweezers, which are placed into the Vitrobot (see Note 5). Blot the
excess media from the grid before adding 3 μL of 20 nm gold
solution and 0.5 μm TetraSpeck Microspheres (see Note 6).
2. We recommend manual blotting from the gold side of the grid
with all Vitrobot parameters set to 0. It is recommended that
the humidity of the Vitrobot chamber be at 95% when the grid
is blotted (see Notes 7 and 8).
3. After plunge-freezing, the grids are stored within grid boxes
cooled in clean liquid nitrogen.
4. Before cryo-LM or cryo-EM imaging, EM grids are clipped
into FEI autogrids.
2.2.5 Cryo-LM Imaging
for Cryo-CLEM Using
the FEI Cryostage
1. In our setup, an FEI cryostage modified to hold Titan Krios
cartridges is used, and liquid nitrogen is continuously pumped
into the stage by a Norton cryo-pump to maintain the temperature at 80 K. Wait approximately 40 min for the stage to
equilibrate and maintain its cryogenic temperature.
2. Load one grid into the stage at a time.
3. Collect a montage or a series of individual phase contrast and
fluorescent Z-stack images of mammalian cells growing flat
within the grid square and near the center of the grid
(Fig. 1). If your target protein is tagged with a green fluorescent protein (GFP) (see Note 9), collection of red, blue, and
green fluorescent images is recommended (see Note 10).
4. Evaluation of ice thickness should be made when imaging
mammalian cells using cryo-LM. Ice cracks and thick ice
around the circumference of the square are indications that
the ice is too thick around the cell. In addition, obvious cracks
in the carbon support close to target cells are not ideal and will
affect the stability of the cell when being tilted, which can lead
to suboptimal tilt-series alignment.
5. Align the fluorescent images to the fluorescent microspheres
observed in the phase contrast image (see Note 11).
88
Mohammed Kaplan et al.