7. Using a micropipette, dispense a 4-μL drop of cell suspension
onto the coverslip surface, in the center of the grease circle (see
Fig. 3b).
8. Using forceps or vacuum tweezers, hold a second 16-mm
circular coverslip 2–3 mm directly above the first coverslip, so
that the two coverslips are parallel to each other and concentrically aligned. Release the top coverslip and allow it to drop onto
the bottom coverslip.
9. If necessary, align the two coverslips to each other by applying
horizontal forces with two sets of angled forceps, as shown in
Fig. 3c–e (see Note 67).
10. Using the tips of a pair of forceps, gently push down on the top
coverslip to simultaneously spread out the liquid sample and
seal the grease ring. Stop when the liquid has spread over a ~1cm
2 area, just making contact with the inner edge of the grease
ring (see Notes 68 and 69).
11. Place the coverslip sandwich onto a Kimwipes tissue. Without
applying excessive pressure (which can squeeze liquid out of
the sample), dry the top and bottom surface of the sample. In
addition, apply the Kimwipes tissue along the entire perimeter
of the coverslip sandwich preparation, to draw out (by capillary
action) any liquid that has escaped the grease barrier (see Note
70).
3.6 Sample Loading
1. Remove the cryomicroscope stage lid, and set it upside-down
onto a clean, dry surface.
2. Use vacuum tweezers to transfer the sample into the cryomicroscope stage chamber, carefully dropping it (from a height
<5 mm) into the G16.3 sample carrier ring on the silver block
of the BCS196 stage (see Note 71).
3. Without delay, start recording of temperature data by clicking
the red triangle in the Data Capture toolbar (see Note 72).
4. Replace the cryomicroscope stage lid, and screw it tight to seal
the chamber.
5. Purge the air from the cryomicroscope stage chamber using
dry nitrogen gas, by completing the procedure described in
steps 6–12 (see Note 73).
6. Manually increase the liquid nitrogen flowrate to its maximum
value (by pressing the upward arrow next to the “Lnp” box in
the Temperature Control Panel, until Lnp ¼ 30) (see Note 74).
7. Insert the supplied gas valve fitting (barbed end facing outward) into the gas port on the back of the BCS196 stage, until
the valve clicks open.
High-Speed Video Cryomicroscopy
235
onto the coverslip surface, in the center of the grease circle (see
Fig. 3b).
8. Using forceps or vacuum tweezers, hold a second 16-mm
circular coverslip 2–3 mm directly above the first coverslip, so
that the two coverslips are parallel to each other and concentrically aligned. Release the top coverslip and allow it to drop onto
the bottom coverslip.
9. If necessary, align the two coverslips to each other by applying
horizontal forces with two sets of angled forceps, as shown in
Fig. 3c–e (see Note 67).
10. Using the tips of a pair of forceps, gently push down on the top
coverslip to simultaneously spread out the liquid sample and
seal the grease ring. Stop when the liquid has spread over a ~1cm
2 area, just making contact with the inner edge of the grease
ring (see Notes 68 and 69).
11. Place the coverslip sandwich onto a Kimwipes tissue. Without
applying excessive pressure (which can squeeze liquid out of
the sample), dry the top and bottom surface of the sample. In
addition, apply the Kimwipes tissue along the entire perimeter
of the coverslip sandwich preparation, to draw out (by capillary
action) any liquid that has escaped the grease barrier (see Note
70).
3.6 Sample Loading
1. Remove the cryomicroscope stage lid, and set it upside-down
onto a clean, dry surface.
2. Use vacuum tweezers to transfer the sample into the cryomicroscope stage chamber, carefully dropping it (from a height
<5 mm) into the G16.3 sample carrier ring on the silver block
of the BCS196 stage (see Note 71).
3. Without delay, start recording of temperature data by clicking
the red triangle in the Data Capture toolbar (see Note 72).
4. Replace the cryomicroscope stage lid, and screw it tight to seal
the chamber.
5. Purge the air from the cryomicroscope stage chamber using
dry nitrogen gas, by completing the procedure described in
steps 6–12 (see Note 73).
6. Manually increase the liquid nitrogen flowrate to its maximum
value (by pressing the upward arrow next to the “Lnp” box in
the Temperature Control Panel, until Lnp ¼ 30) (see Note 74).
7. Insert the supplied gas valve fitting (barbed end facing outward) into the gas port on the back of the BCS196 stage, until
the valve clicks open.
High-Speed Video Cryomicroscopy
235