64. The dispensing needle should be held perpendicular to the
glass surface. Hold the coverslip steady by applying a downward force using the forceps.
65. The total amount of silicone grease transferred to the coverslip
surface should be approximately 2 mg.
66. Small gaps in the circle perimeter are acceptable. The sample
does not need to be hermetically sealed. The purpose of the
grease ring is fourfold: (1) to provide adhesion to hold the
coverslip sandwich construct together; (2) to reduce the risk of
leakage of the liquid sample onto the cryomicroscope silver
block; (3) to control the thickness of the liquid sample; and
(4) to reduce the rate of evaporation of water from the sample.
67. Do not push down on the coverslips before they have been
aligned. Hold the two angled tips of the first pair of forceps
against the work surface, approximately 1 cm apart and perpendicular to the surface; this pair of tips will serve as a brace
for the bottom coverslip. Gently nudge the bottom coverslip
so that its edge makes contact with both tips of the bracing
forceps, while the overhang of the misaligned top coverslip
should be oriented to be diametrically opposed to the bracing
forceps. In a similar manner, the second set of forceps can be
held with its angled tips perpendicular to the work surface and
brought into contact with the top coverslip overhang (i.e., on
the side opposite the first set of forceps), as shown in Fig. 3c, d.
By carefully moving the two sets of forceps toward each other,
the two coverslips will be aligned within the square-shaped
arrangement of the four tips (see Fig. 3e).
68. For best results, push only on the part of the coverslip that is
directly above the grease ring, by running the tip of the forceps
along the perimeter of the grease ring while applying gentle
pressure. Observe the response of the grease and the aqueous
sample during this process. The grease should spread to a width
of 1–2 mm, but there will typically be significant variations in
width along the perimeter of the grease circle (thus, it is
important to ensure that the silicone grease never spreads so
far as to reach the edge of the coverslip). Simultaneously, the
liquid sample will spread out due to the capillary forces that
arise when the two coverslips are brought closer together.
Although it is normal for the aqueous liquid to occasionally
breach the grease barrier, one should attempt to minimize such
leaks by removing all pressure when the liquid reaches the
inside edge of the grease ring. At no time should any liquid
be squeezed out of the coverslip sandwich (i.e., past the coverslip edge). If necessary, one may use a slightly smaller volume of
liquid in step 7 of Subheading 3.5 (e.g., ~3 μL), although this
High-Speed Video Cryomicroscopy
259
glass surface. Hold the coverslip steady by applying a downward force using the forceps.
65. The total amount of silicone grease transferred to the coverslip
surface should be approximately 2 mg.
66. Small gaps in the circle perimeter are acceptable. The sample
does not need to be hermetically sealed. The purpose of the
grease ring is fourfold: (1) to provide adhesion to hold the
coverslip sandwich construct together; (2) to reduce the risk of
leakage of the liquid sample onto the cryomicroscope silver
block; (3) to control the thickness of the liquid sample; and
(4) to reduce the rate of evaporation of water from the sample.
67. Do not push down on the coverslips before they have been
aligned. Hold the two angled tips of the first pair of forceps
against the work surface, approximately 1 cm apart and perpendicular to the surface; this pair of tips will serve as a brace
for the bottom coverslip. Gently nudge the bottom coverslip
so that its edge makes contact with both tips of the bracing
forceps, while the overhang of the misaligned top coverslip
should be oriented to be diametrically opposed to the bracing
forceps. In a similar manner, the second set of forceps can be
held with its angled tips perpendicular to the work surface and
brought into contact with the top coverslip overhang (i.e., on
the side opposite the first set of forceps), as shown in Fig. 3c, d.
By carefully moving the two sets of forceps toward each other,
the two coverslips will be aligned within the square-shaped
arrangement of the four tips (see Fig. 3e).
68. For best results, push only on the part of the coverslip that is
directly above the grease ring, by running the tip of the forceps
along the perimeter of the grease ring while applying gentle
pressure. Observe the response of the grease and the aqueous
sample during this process. The grease should spread to a width
of 1–2 mm, but there will typically be significant variations in
width along the perimeter of the grease circle (thus, it is
important to ensure that the silicone grease never spreads so
far as to reach the edge of the coverslip). Simultaneously, the
liquid sample will spread out due to the capillary forces that
arise when the two coverslips are brought closer together.
Although it is normal for the aqueous liquid to occasionally
breach the grease barrier, one should attempt to minimize such
leaks by removing all pressure when the liquid reaches the
inside edge of the grease ring. At no time should any liquid
be squeezed out of the coverslip sandwich (i.e., past the coverslip edge). If necessary, one may use a slightly smaller volume of
liquid in step 7 of Subheading 3.5 (e.g., ~3 μL), although this
High-Speed Video Cryomicroscopy
259