2.1 Common
Materials
and Equipment
1. The EM grid, a universal, and versatile specimen support. The
universal support to manipulate EM samples is a thin metallic
grid 3 mm in diameter adapted for most electron microscope
holders. EM grids are made of copper, copper/rhodium, or
gold to be conductive and eliminate electron charge accumulation. Square holes of different sizes are separated by grid bars
that absorb electrons. Copper mesh grids with 300 grid squares
per inch are commonly used because they are conductive, stable
under the electron beam, and inexpensive. The grid is generally
covered by a 5–20 nm thin carbon film onto which protein
complexes are adsorbed for subsequent negative staining. To
avoid sample adsorption in cryo-EM, the grids can also be
covered with a self-made carbon film perforated with holes of
various diameter ranging between 0.3 and 10 μm in diameter
[7]. More recently, perforated grids with calibrated circular
holes arranged into regular arrays were developed to serve
automated data acquisition [8]. Such perforated grids can be
covered by a continuous thin carbon film when adsorption
cannot be avoided. Holey carbon films can also be covered by
a single monolayer thick graphene or graphene oxide crystalline layer instead of amorphous carbon, thus leading to a less
grainy background [9] (Oxford Instruments, quantifoil micro
tools GMBH).
2. High precision tweezers. To properly manipulate the EM grids
without distortion and contamination, high precision and
sharp tweezers are recommended (Dumont style 5 tweezers,
Fine Science Tools GMBH ref.: 11252-30). Make sure to grab
the grids by the rim to not damage or distort the support.
Standard straight tweezers are sufficient to manipulate grids,
but other types of tweezers can be useful in electron microscopy: anti-capillarity tweezers have a wider angle at the tip
when closed, limiting the amount of liquid retained by capillarity. Inverted tweezers are also found useful since they remain
closed until squeezed, limiting the risks of dropping grids when
handling the tweezers.
3. Glow discharge and plasma cleaner devices. These table top
instruments are used to produce a gas plasma to charge the
surface of the grids by an electrostatic potential in order to
produce a hydrophilic surface suitable for protein absorption.
The devices differ by the quality of the vacuum (between 10
À2
and 10
À6 Torr, or 1.3 and 1.3 Â 10
À4 Pa), the way the plasma is
produced, and the mixture of gases that can be used to form
the plasma (ELMO, Cordouan Technologies; Nanoclean
model 1070, Fischione).
4. Filter paper. Different steps of the protocol require blotting the
grids to remove excess of staining solution or water (MachereyNagel, ref.: 202009).
246
Alexandre Frechard et al.
Materials
and Equipment
1. The EM grid, a universal, and versatile specimen support. The
universal support to manipulate EM samples is a thin metallic
grid 3 mm in diameter adapted for most electron microscope
holders. EM grids are made of copper, copper/rhodium, or
gold to be conductive and eliminate electron charge accumulation. Square holes of different sizes are separated by grid bars
that absorb electrons. Copper mesh grids with 300 grid squares
per inch are commonly used because they are conductive, stable
under the electron beam, and inexpensive. The grid is generally
covered by a 5–20 nm thin carbon film onto which protein
complexes are adsorbed for subsequent negative staining. To
avoid sample adsorption in cryo-EM, the grids can also be
covered with a self-made carbon film perforated with holes of
various diameter ranging between 0.3 and 10 μm in diameter
[7]. More recently, perforated grids with calibrated circular
holes arranged into regular arrays were developed to serve
automated data acquisition [8]. Such perforated grids can be
covered by a continuous thin carbon film when adsorption
cannot be avoided. Holey carbon films can also be covered by
a single monolayer thick graphene or graphene oxide crystalline layer instead of amorphous carbon, thus leading to a less
grainy background [9] (Oxford Instruments, quantifoil micro
tools GMBH).
2. High precision tweezers. To properly manipulate the EM grids
without distortion and contamination, high precision and
sharp tweezers are recommended (Dumont style 5 tweezers,
Fine Science Tools GMBH ref.: 11252-30). Make sure to grab
the grids by the rim to not damage or distort the support.
Standard straight tweezers are sufficient to manipulate grids,
but other types of tweezers can be useful in electron microscopy: anti-capillarity tweezers have a wider angle at the tip
when closed, limiting the amount of liquid retained by capillarity. Inverted tweezers are also found useful since they remain
closed until squeezed, limiting the risks of dropping grids when
handling the tweezers.
3. Glow discharge and plasma cleaner devices. These table top
instruments are used to produce a gas plasma to charge the
surface of the grids by an electrostatic potential in order to
produce a hydrophilic surface suitable for protein absorption.
The devices differ by the quality of the vacuum (between 10
À2
and 10
À6 Torr, or 1.3 and 1.3 Â 10
À4 Pa), the way the plasma is
produced, and the mixture of gases that can be used to form
the plasma (ELMO, Cordouan Technologies; Nanoclean
model 1070, Fischione).
4. Filter paper. Different steps of the protocol require blotting the
grids to remove excess of staining solution or water (MachereyNagel, ref.: 202009).
246
Alexandre Frechard et al.