3.1.4 Cryo-CLEM
Correlated light and electron microscopy (CLEM) of the same
specimen is a powerful technique that allows targeting labeled
cellular structures of interest for high-resolution EM
[28, 36]. Cryo-CLEM combines the advantage of fluorescence
microscopy to specifically locate organelles or proteins of interest
and the advantage of TEM to visualize the same object at high
resolution in its native cellular context. A full description of
CLEM is outside the scope of this chapter. For FIB milling, correlation can provide great advantages in determining areas to mill,
but also provides additional challenges for sample preparation
including reduced throughput and increased atmospheric ice contamination due to additional imaging and transfer steps.
For typical mammalian and yeast samples, the cell density
described above (about 1–2 cells or clumps per grid square) is
typically compatible with cryo-fluorescent microscopy. For bacterial specimens, the cell density should be reduced to somewhat less
than full coverage of the grid square in order to be able to resolve
individual cells.
During FIB milling, the fluorescent data can be used to guide
targeted milling of mammalian and yeast samples. For bacteria,
milling is still done in non-targeted manner—while the fluorescent
information is useful to screen individual grid squares for suitable
cell coverage, ice thickness, and overall quality, the fluorescent data
is more critical at the TEM to determine which specific cells should
be targeted for tilt series acquisition.
There are commercially available cryo-fluorescence microscopes such as the Corrsight and the Leica Cryo-CLEM system.
Additionally, aftermarket cryo-stage additions such as CMS-196
Linkam stage are available in addition to a number of custommade cryo-light microscopes [37, 38]. Recent work has also
demonstrated cryo super-resolution microscopy to be compatible
with a typical cryo-CLEM workflow [28, 37, 39–42]. Key points to
consider when acquiring cryo-fluorescence microscopes include
stage temperature stability, anti-contamination features, and compatibility with existing EM instruments and workflows.
3.2 General FIB
Milling Protocol
3.2.1 Before Starting
These steps should be done before the FIB session.
1. Culture and plunge-freeze specimens at the appropriate density
(see Subheading 3.1) using your selected grid type (see Note 2).
Cells should be frozen on the carbon side of quantifoil grids.
2. We recommend marking the autogrids with permanent marker
before clipping to make sample more visible when under liquid
nitrogen (Fig. 2a). Avoid marking the milling notch of the
cryo-FIB-autogrid due to potential beam interactions with
the marker residue.
62
Vinson Lam and Elizabeth Villa
Correlated light and electron microscopy (CLEM) of the same
specimen is a powerful technique that allows targeting labeled
cellular structures of interest for high-resolution EM
[28, 36]. Cryo-CLEM combines the advantage of fluorescence
microscopy to specifically locate organelles or proteins of interest
and the advantage of TEM to visualize the same object at high
resolution in its native cellular context. A full description of
CLEM is outside the scope of this chapter. For FIB milling, correlation can provide great advantages in determining areas to mill,
but also provides additional challenges for sample preparation
including reduced throughput and increased atmospheric ice contamination due to additional imaging and transfer steps.
For typical mammalian and yeast samples, the cell density
described above (about 1–2 cells or clumps per grid square) is
typically compatible with cryo-fluorescent microscopy. For bacterial specimens, the cell density should be reduced to somewhat less
than full coverage of the grid square in order to be able to resolve
individual cells.
During FIB milling, the fluorescent data can be used to guide
targeted milling of mammalian and yeast samples. For bacteria,
milling is still done in non-targeted manner—while the fluorescent
information is useful to screen individual grid squares for suitable
cell coverage, ice thickness, and overall quality, the fluorescent data
is more critical at the TEM to determine which specific cells should
be targeted for tilt series acquisition.
There are commercially available cryo-fluorescence microscopes such as the Corrsight and the Leica Cryo-CLEM system.
Additionally, aftermarket cryo-stage additions such as CMS-196
Linkam stage are available in addition to a number of custommade cryo-light microscopes [37, 38]. Recent work has also
demonstrated cryo super-resolution microscopy to be compatible
with a typical cryo-CLEM workflow [28, 37, 39–42]. Key points to
consider when acquiring cryo-fluorescence microscopes include
stage temperature stability, anti-contamination features, and compatibility with existing EM instruments and workflows.
3.2 General FIB
Milling Protocol
3.2.1 Before Starting
These steps should be done before the FIB session.
1. Culture and plunge-freeze specimens at the appropriate density
(see Subheading 3.1) using your selected grid type (see Note 2).
Cells should be frozen on the carbon side of quantifoil grids.
2. We recommend marking the autogrids with permanent marker
before clipping to make sample more visible when under liquid
nitrogen (Fig. 2a). Avoid marking the milling notch of the
cryo-FIB-autogrid due to potential beam interactions with
the marker residue.
62
Vinson Lam and Elizabeth Villa