1. Liquid nitrogen. Liquid nitrogen is used at several steps in the
protocol to refrigerate and liquefy ethane, to store the vitrified
grid and cryo-box, and to transfer the cryo-box to a dedicated
short-term or long-term storage device. Once vitrified, the
specimen has to be kept at liquid nitrogen temperature (boiling
temperature of À196
C under normal atmospheric pressure)
to avoid water devitrification.
2. Plastic beaker. A 1 L polypropylene beaker with a handle to
pour nitrogen (Vitlab, ref.: 442941).
3. Cryo-boxes. These round boxes are used to store the vitrified
samples in liquid nitrogen. They generally hold four EM grids
and have a transparent cover hold in place by a screw. The cover
has a single slit and can be rotated to select one of the four EM
grid slots (Oxford Instruments, ref.: G3539).
4. Screw driver. A screw driver is necessary to fasten and unfasten
the cryo-box cover immersed in liquid nitrogen.
5. Long isolated tweezers. Twenty-centimeter long tweezers are
used to manipulate the cryo-boxes submerged in nitrogen. To
protect the operator from frostbites the tweezers should be
cold-isolated by a protective layer.
6. Liquid nitrogen tank. A 5 L liquid nitrogen container is
required to store over longer periods the cryo-boxes which
contain the grids. The cryo-boxes can be put into Falcon
tubes perforated for liquid nitrogen influx and fixed to a
2–3 mm thick plastic-coated metallic cord which hangs out of
the tank once the tube is immersed. More elaborate systems
have been developed to organize multi-user grid storage, efficient cryo-box retrieval, and automated nitrogen refill (EMS
Cryo Pucks G2).
7. Cryo-plunger. Specimen vitrification is commonly achieved by
forming a thin layer of the sample on the EM grid through
blotting away the excess of sample and subsequent plunging of
the grid into a liquid ethane slush refrigerated by liquid nitrogen [5]. Simple, versatile, and portable devices were designed
in which a tweezer holding the grid was fixed on a weighted
friction-reduced rod that moved by gravity to plunge the grid
into the cryogen container. Ethane is liquefied in a 2 mL
aluminum container inserted in a styrofoam container of liquid
nitrogen. Since the boiling temperature of liquid nitrogen is
lower than the melting temperature of ethane (À183.3
C), the
ethane will tend to solidify. An ethane heating device or a
properly designed ethane tank assures that ethane does not
reach liquid nitrogen temperature and stays as a liquid slush.
Highly automated devices are currently available, providing a
temperature and humidity controlled chamber to avoid
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Alexandre Frechard et al.
protocol to refrigerate and liquefy ethane, to store the vitrified
grid and cryo-box, and to transfer the cryo-box to a dedicated
short-term or long-term storage device. Once vitrified, the
specimen has to be kept at liquid nitrogen temperature (boiling
temperature of À196
C under normal atmospheric pressure)
to avoid water devitrification.
2. Plastic beaker. A 1 L polypropylene beaker with a handle to
pour nitrogen (Vitlab, ref.: 442941).
3. Cryo-boxes. These round boxes are used to store the vitrified
samples in liquid nitrogen. They generally hold four EM grids
and have a transparent cover hold in place by a screw. The cover
has a single slit and can be rotated to select one of the four EM
grid slots (Oxford Instruments, ref.: G3539).
4. Screw driver. A screw driver is necessary to fasten and unfasten
the cryo-box cover immersed in liquid nitrogen.
5. Long isolated tweezers. Twenty-centimeter long tweezers are
used to manipulate the cryo-boxes submerged in nitrogen. To
protect the operator from frostbites the tweezers should be
cold-isolated by a protective layer.
6. Liquid nitrogen tank. A 5 L liquid nitrogen container is
required to store over longer periods the cryo-boxes which
contain the grids. The cryo-boxes can be put into Falcon
tubes perforated for liquid nitrogen influx and fixed to a
2–3 mm thick plastic-coated metallic cord which hangs out of
the tank once the tube is immersed. More elaborate systems
have been developed to organize multi-user grid storage, efficient cryo-box retrieval, and automated nitrogen refill (EMS
Cryo Pucks G2).
7. Cryo-plunger. Specimen vitrification is commonly achieved by
forming a thin layer of the sample on the EM grid through
blotting away the excess of sample and subsequent plunging of
the grid into a liquid ethane slush refrigerated by liquid nitrogen [5]. Simple, versatile, and portable devices were designed
in which a tweezer holding the grid was fixed on a weighted
friction-reduced rod that moved by gravity to plunge the grid
into the cryogen container. Ethane is liquefied in a 2 mL
aluminum container inserted in a styrofoam container of liquid
nitrogen. Since the boiling temperature of liquid nitrogen is
lower than the melting temperature of ethane (À183.3
C), the
ethane will tend to solidify. An ethane heating device or a
properly designed ethane tank assures that ethane does not
reach liquid nitrogen temperature and stays as a liquid slush.
Highly automated devices are currently available, providing a
temperature and humidity controlled chamber to avoid
248
Alexandre Frechard et al.