specimen. While sectioning is possible (see below), a number of other options
should first be considered. It may be possible to choose an alternative, thinner,
species such as imaging Ostreococcus tauri (the smallest known eukaryote) to
study the eukaryotic spindle [17]. If the organism is genetically manipulable it may
be possible to make it thinner, as has recently been done successfully with bacterial
‘minicells’ [18] or thinner cells [19, 20]. Different media may also lead cells to
grow thinner than in standard media [21]. If these non-disruptive approaches fail,
thick cells can be gently deflated without full lysis. For example, the bacterial cell
wall can be punctured using lysozyme or penicillin to reduce the thickness of the
still quasi-intact cell [22]. Partial purification of intact organelles can be achieved as
illustrated by purification of eukaryotic mitochondria [23], nuclei [24], or bacterial
chemoreceptor arrays [25].
3.3.1 Vitrifying Thin Specimens: Plunge-Freezing
Thin specimens (up to a few microns) can be vitrified by direct plunging into a
liquid cryogen. The cooling rate is lower for thicker specimens, which increases the
incidence of crystalline ice that damages biological material and diffracts electrons.
Specimens are typically mixed with 5–20 nm-diameter gold fiducial markers for tilt
series alignment, and applied to a standard 3 mm-diameter electron microscopy
grid overlayed with a thin support film. The support film is commonly carbon,
although gold support provides stability advantages [26]. Support films are typically glow discharged to make them hydrophilic for specimen adhesion.
A number of cryogens can be used for vitrification. The low heat of vaporization
and low boiling point (77 K) of nitrogen means it vaporizes to form an insulating
gaseous envelope around the specimen, rendering it unsuitable as a cryogen. More
appropriate cryogens with higher boiling points and greater heat capacities can be
cooled in a metal cup within an outer reservoir of liquid nitrogen. Ethane’s greater
heat capacity and high boiling point (184.5 K) make it widely-used. Ethane cooled
by liquid nitrogen freezes at 90 K, however, requiring the user to supply heat
occasionally to keep the ethane liquid yet cold enough to vitrify. A mixture of
ethane with propane conveniently avoids this problem since the mixture remains
liquid down to 77 K [27].
To achieve suspension of the specimen in a very thin aqueous envelope for
vitrification, specimen suspension is conventionally applied to the grid with a pipette, although it is also possible to dip the grid into the suspension. The specimen
accumulates in holes in the grid support film, and excess liquid is removed by
blotting with filter paper to leave a thin envelope. Blotting is achieved either manually by carefully touching a piece of filter paper to the grid held by a homemade
‘gravity plunger’ device that can be triggered to plunge into cryogen (the grid held in
tweezers on the end of a vertical rod), or by using a commercial device that uses
motorized blotting pads inside a humidity- and temperature-controlled chamber,
together with motorized or pneumatic plunging. Such commercial devices allow for
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J. L. Ferreira et al.
should first be considered. It may be possible to choose an alternative, thinner,
species such as imaging Ostreococcus tauri (the smallest known eukaryote) to
study the eukaryotic spindle [17]. If the organism is genetically manipulable it may
be possible to make it thinner, as has recently been done successfully with bacterial
‘minicells’ [18] or thinner cells [19, 20]. Different media may also lead cells to
grow thinner than in standard media [21]. If these non-disruptive approaches fail,
thick cells can be gently deflated without full lysis. For example, the bacterial cell
wall can be punctured using lysozyme or penicillin to reduce the thickness of the
still quasi-intact cell [22]. Partial purification of intact organelles can be achieved as
illustrated by purification of eukaryotic mitochondria [23], nuclei [24], or bacterial
chemoreceptor arrays [25].
3.3.1 Vitrifying Thin Specimens: Plunge-Freezing
Thin specimens (up to a few microns) can be vitrified by direct plunging into a
liquid cryogen. The cooling rate is lower for thicker specimens, which increases the
incidence of crystalline ice that damages biological material and diffracts electrons.
Specimens are typically mixed with 5–20 nm-diameter gold fiducial markers for tilt
series alignment, and applied to a standard 3 mm-diameter electron microscopy
grid overlayed with a thin support film. The support film is commonly carbon,
although gold support provides stability advantages [26]. Support films are typically glow discharged to make them hydrophilic for specimen adhesion.
A number of cryogens can be used for vitrification. The low heat of vaporization
and low boiling point (77 K) of nitrogen means it vaporizes to form an insulating
gaseous envelope around the specimen, rendering it unsuitable as a cryogen. More
appropriate cryogens with higher boiling points and greater heat capacities can be
cooled in a metal cup within an outer reservoir of liquid nitrogen. Ethane’s greater
heat capacity and high boiling point (184.5 K) make it widely-used. Ethane cooled
by liquid nitrogen freezes at 90 K, however, requiring the user to supply heat
occasionally to keep the ethane liquid yet cold enough to vitrify. A mixture of
ethane with propane conveniently avoids this problem since the mixture remains
liquid down to 77 K [27].
To achieve suspension of the specimen in a very thin aqueous envelope for
vitrification, specimen suspension is conventionally applied to the grid with a pipette, although it is also possible to dip the grid into the suspension. The specimen
accumulates in holes in the grid support film, and excess liquid is removed by
blotting with filter paper to leave a thin envelope. Blotting is achieved either manually by carefully touching a piece of filter paper to the grid held by a homemade
‘gravity plunger’ device that can be triggered to plunge into cryogen (the grid held in
tweezers on the end of a vertical rod), or by using a commercial device that uses
motorized blotting pads inside a humidity- and temperature-controlled chamber,
together with motorized or pneumatic plunging. Such commercial devices allow for
66
J. L. Ferreira et al.