R2/2 square supports coated with Quantifoil holey carbon film
are recommended. The R2/2 square support allows a sufficient
area for the cells to adequately attach and spread on the grid.
4. To prevent the grid from sliding on the glass slide, both sides of
the grid are coated with collagen.
5. We recommend touching the grid to the edge of the glass slide
to gain optimal purchase.
6. Due to their increased contrast in low signal:noise tomograms
of mammalian cells, 20 nm gold beads are used as fiducial
markers to align the tilt-series. If performing cryo-CLEM,
fluorescent microspheres are mixed and vortexed in 1:1 dilution with the 20 nm gold beads before being added to the grid.
It is recommended that the gold beads be concentrated as
much possible to ensure their presence around the thin edges
of cells.
7. The grid is blotted from the side where the cells are not
attached so as to prevent damaging the cells during the blotting
process. Blotting is achieved by using a strip of blotting paper
which has been folded 90
at its tip and held by tweezers which
enter the Vitrobot chamber from the side port.
8. A liquid ethane/propane mixture is used to avoid solidification
as when using ethane alone.
9. In our experience, the signal from GFP can be too weak to
detect due to high background autofluorescence in cryogenic
conditions. Using the brighter mNeonGreen fluorescent protein is recommended.
10. Collection of phase contrast and red, green, and blue fluorescence images is needed to distinguish autofluorescence from
real fluorescent protein signal [53]. For example, in U2OS cells
the blue channel can be used to distinguish the 0.5 μm TetraSpeck Microspheres (which fluoresce bright red, green and
blue, and are seen by phase contrast) from autofluorescence
(bright red and green, dim blue), and real green fluorescent
protein signal (bright green). We recommend plotting the
intensities of puncta in the red, green, and blue channels in
unlabeled cells vs. cells expressing the fluorescent protein to
identify targets.
11. Aligning the fluorescent images to the fluorescent microspheres seen in the phase contrast image can help identify
whether the target fluorescent protein signal is present in an
area of the ice that has sufficient electron transparency.
12. Mixing HCl and bleach creates very hazardous chlorine gas
therefore this should be done in a chemical fumehood with an
exhaust leading to the exterior of the building rather than a
hood which recycles air in the same room.
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Mohammed Kaplan et al.
are recommended. The R2/2 square support allows a sufficient
area for the cells to adequately attach and spread on the grid.
4. To prevent the grid from sliding on the glass slide, both sides of
the grid are coated with collagen.
5. We recommend touching the grid to the edge of the glass slide
to gain optimal purchase.
6. Due to their increased contrast in low signal:noise tomograms
of mammalian cells, 20 nm gold beads are used as fiducial
markers to align the tilt-series. If performing cryo-CLEM,
fluorescent microspheres are mixed and vortexed in 1:1 dilution with the 20 nm gold beads before being added to the grid.
It is recommended that the gold beads be concentrated as
much possible to ensure their presence around the thin edges
of cells.
7. The grid is blotted from the side where the cells are not
attached so as to prevent damaging the cells during the blotting
process. Blotting is achieved by using a strip of blotting paper
which has been folded 90
at its tip and held by tweezers which
enter the Vitrobot chamber from the side port.
8. A liquid ethane/propane mixture is used to avoid solidification
as when using ethane alone.
9. In our experience, the signal from GFP can be too weak to
detect due to high background autofluorescence in cryogenic
conditions. Using the brighter mNeonGreen fluorescent protein is recommended.
10. Collection of phase contrast and red, green, and blue fluorescence images is needed to distinguish autofluorescence from
real fluorescent protein signal [53]. For example, in U2OS cells
the blue channel can be used to distinguish the 0.5 μm TetraSpeck Microspheres (which fluoresce bright red, green and
blue, and are seen by phase contrast) from autofluorescence
(bright red and green, dim blue), and real green fluorescent
protein signal (bright green). We recommend plotting the
intensities of puncta in the red, green, and blue channels in
unlabeled cells vs. cells expressing the fluorescent protein to
identify targets.
11. Aligning the fluorescent images to the fluorescent microspheres seen in the phase contrast image can help identify
whether the target fluorescent protein signal is present in an
area of the ice that has sufficient electron transparency.
12. Mixing HCl and bleach creates very hazardous chlorine gas
therefore this should be done in a chemical fumehood with an
exhaust leading to the exterior of the building rather than a
hood which recycles air in the same room.
108
Mohammed Kaplan et al.