cryoprotectant additives, etc. In practice, intracellular freezing
events may occur at stage temperatures lower than the
expected minimum nucleation temperature, as a result of unanticipated cell dehydration or vertical temperature gradients.
Thus, an additional margin of 5–10
C past the lowest
expected intracellular ice formation temperature is advisable.
46. Data analysis will be greatly simplified if the duration of the
video recording is longer than the duration of the freezing
ramp (the cooling step in Ramp 4) but shorter than the total
duration of Ramp 4 (including the cooling step and the isothermal hold period). Thus, the data analysis approach
described in Subheading 3.9 assumes that the warming ramp
(Ramp 5) does not begin until after the video recording has
ended. To ensure that the video recording does not overlap
the warming ramp, it may be necessary to adjust the length of
the isothermal hold at the end of the cooling step, to extend
the total duration of Ramp 4. For example, in Table 1, the
duration of the rapid-freezing ramp is 79.6 s (including the
specified 1-min hold at À50
C). Thus, if recording 8-bit video
at a resolution of 300 Â 300 pixels and a frame rate of 2000 fps,
a camera memory capacity of 4 GB will yield 23.9 s of recording time; for the temperature profile specified in Table 1, this
recording duration is sufficient to capture the cooling from À1
to À50
C (19.6 s), whereas the video recording will have
terminated well before the start of the warming ramp. Conversely, without the 1-min hold time at À50
C, the last ~2500
frames of the video recording would depict the warming
response from À50
C to approximately À47
C (i.e., the
initial part of Ramp 5).
47. After entering all values in the temperature profile table, save
the profile by selecting “Save,” and then “Temperature Profile,” under the “File” menu. Specify the desired location and
name of the temperature profile file, and then click the “Save”
button. The protocols described in Subheadings 3.6 and 3.7
assume that the saved temperature profile starts and ends with
an indefinite hold at the sample loading temperature (e.g.,
Ramp 1 and Ramp 5 in Table 1).
48. This will reduce the accumulation of frost on the coolant inlet
pipe inside the BCS196 chamber. Any water allowed to accumulate within the chamber may make the purging procedure
less effective.
49. Clearance can be further improved by rotating the objective
turret so that the shortest objective (or an empty objective
position) faces the front. If the BCS196 mounting allows for
translational (XY) movement of the cryomicroscope stage
254
Jens O. M. Karlsson
events may occur at stage temperatures lower than the
expected minimum nucleation temperature, as a result of unanticipated cell dehydration or vertical temperature gradients.
Thus, an additional margin of 5–10
C past the lowest
expected intracellular ice formation temperature is advisable.
46. Data analysis will be greatly simplified if the duration of the
video recording is longer than the duration of the freezing
ramp (the cooling step in Ramp 4) but shorter than the total
duration of Ramp 4 (including the cooling step and the isothermal hold period). Thus, the data analysis approach
described in Subheading 3.9 assumes that the warming ramp
(Ramp 5) does not begin until after the video recording has
ended. To ensure that the video recording does not overlap
the warming ramp, it may be necessary to adjust the length of
the isothermal hold at the end of the cooling step, to extend
the total duration of Ramp 4. For example, in Table 1, the
duration of the rapid-freezing ramp is 79.6 s (including the
specified 1-min hold at À50
C). Thus, if recording 8-bit video
at a resolution of 300 Â 300 pixels and a frame rate of 2000 fps,
a camera memory capacity of 4 GB will yield 23.9 s of recording time; for the temperature profile specified in Table 1, this
recording duration is sufficient to capture the cooling from À1
to À50
C (19.6 s), whereas the video recording will have
terminated well before the start of the warming ramp. Conversely, without the 1-min hold time at À50
C, the last ~2500
frames of the video recording would depict the warming
response from À50
C to approximately À47
C (i.e., the
initial part of Ramp 5).
47. After entering all values in the temperature profile table, save
the profile by selecting “Save,” and then “Temperature Profile,” under the “File” menu. Specify the desired location and
name of the temperature profile file, and then click the “Save”
button. The protocols described in Subheadings 3.6 and 3.7
assume that the saved temperature profile starts and ends with
an indefinite hold at the sample loading temperature (e.g.,
Ramp 1 and Ramp 5 in Table 1).
48. This will reduce the accumulation of frost on the coolant inlet
pipe inside the BCS196 chamber. Any water allowed to accumulate within the chamber may make the purging procedure
less effective.
49. Clearance can be further improved by rotating the objective
turret so that the shortest objective (or an empty objective
position) faces the front. If the BCS196 mounting allows for
translational (XY) movement of the cryomicroscope stage
254
Jens O. M. Karlsson