shorter than the duration of the cooling portion of the ramp,
one should decrease the image acquisition rate or the ROI
dimensions to increase the available recording duration (see
Note 58).
9. Confirm that the end of the video recording will occur before
the start of the warming ramp in the temperature profile (Ramp
5 in Table 1). To do so, compute the total duration of the
preceding rapid-cooling ramp and isothermal hold (Ramp 4 in
Table 1), by taking the difference between the ramp’s start and
end temperatures, dividing this difference by the cooling rate,
then adding any hold time specified for Ramp 4, and finally,
multiplying the resulting value by the conversion factor 60 s/
min (to convert the ramp duration to seconds). The total
duration thus calculated should be at least ~1 s longer than
the post-trigger recording duration (from step 7 above). If
necessary, increase the image acquisition rate or the ROI
dimensions to decrease the total recording duration, or
increase the hold time associated with Ramp 4 (Table 1) to
extend the ramp duration (see Note 59).
10. Iteratively repeat steps 7–9 above until the video recording
interval is properly matched to the duration of the rapidcooling ramp.
11. Save the final camera configuration as a camera setup file (see
Note 60).
3.5 Sample
Preparation
1. Create a grease dispenser by removing the plunger from a
5-mL Luer-lock syringe, dispensing ~2 mL silicone grease
into the syringe barrel, and then reinserting the plunger into
the barrel (see Note 61). Attach a blunt-tipped Luer-lock dispensing needle (see Notes 62 and 63).
2. Place a 16-mm diameter circular coverslip onto a clean flat
surface, and stabilize it by applying a downward force using
the tips of a clean pair of angled forceps.
3. Push the syringe plunger until a small amount (<0.5 mm) of
grease protrudes from the tip of the dispensing needle.
4. Apply a small (<0.5 mm in diameter) dot of silicone grease
approximately 2 mm away from the coverslip edge, by daubing
the surface of the coverslip with the dispensing needle tip (see
Note 64).
5. Continue to apply a total of approximately 20 such silicone
grease daubs to the coverslip, evenly spaced along a circle
~12 mm in diameter (always ~2 mm away from the coverslip
edge), as shown in Fig. 3a. Apply pressure to the syringe
plunger to advance more silicone grease as necessary (see
Note 65).
High-Speed Video Cryomicroscopy
233
one should decrease the image acquisition rate or the ROI
dimensions to increase the available recording duration (see
Note 58).
9. Confirm that the end of the video recording will occur before
the start of the warming ramp in the temperature profile (Ramp
5 in Table 1). To do so, compute the total duration of the
preceding rapid-cooling ramp and isothermal hold (Ramp 4 in
Table 1), by taking the difference between the ramp’s start and
end temperatures, dividing this difference by the cooling rate,
then adding any hold time specified for Ramp 4, and finally,
multiplying the resulting value by the conversion factor 60 s/
min (to convert the ramp duration to seconds). The total
duration thus calculated should be at least ~1 s longer than
the post-trigger recording duration (from step 7 above). If
necessary, increase the image acquisition rate or the ROI
dimensions to decrease the total recording duration, or
increase the hold time associated with Ramp 4 (Table 1) to
extend the ramp duration (see Note 59).
10. Iteratively repeat steps 7–9 above until the video recording
interval is properly matched to the duration of the rapidcooling ramp.
11. Save the final camera configuration as a camera setup file (see
Note 60).
3.5 Sample
Preparation
1. Create a grease dispenser by removing the plunger from a
5-mL Luer-lock syringe, dispensing ~2 mL silicone grease
into the syringe barrel, and then reinserting the plunger into
the barrel (see Note 61). Attach a blunt-tipped Luer-lock dispensing needle (see Notes 62 and 63).
2. Place a 16-mm diameter circular coverslip onto a clean flat
surface, and stabilize it by applying a downward force using
the tips of a clean pair of angled forceps.
3. Push the syringe plunger until a small amount (<0.5 mm) of
grease protrudes from the tip of the dispensing needle.
4. Apply a small (<0.5 mm in diameter) dot of silicone grease
approximately 2 mm away from the coverslip edge, by daubing
the surface of the coverslip with the dispensing needle tip (see
Note 64).
5. Continue to apply a total of approximately 20 such silicone
grease daubs to the coverslip, evenly spaced along a circle
~12 mm in diameter (always ~2 mm away from the coverslip
edge), as shown in Fig. 3a. Apply pressure to the syringe
plunger to advance more silicone grease as necessary (see
Note 65).
High-Speed Video Cryomicroscopy
233