may result in air gaps within the grease circle (which are also
undesirable).
69. The techniques described here will yield a coverslip sandwich in
which the thickness of the liquid sample is approximately
30–50 μm. This sample thickness is appropriate for cells with
diameters in the range 10–30 μm. If necessary, the sample
thickness can be varied by altering the amount of grease or
the liquid sample volume. However, the sample thickness
should be kept as small as possible without deforming the cells.
70. It is important that the surfaces and edges of the sample be
completely dry before it is loaded into the cryomicroscope
stage. Residual liquid on the sample can cause serious problems, including contamination of the silver block surface
(which can affect temperature control), capillary adhesion of
the bottom coverslip to the silver block (which may cause the
sandwich construct to delaminate when attempting to move
the sample), and ice formation on the external surfaces of the
coverslips (which will cause the view of the sample to be
obscured, or result in confounding optical interference that
can be misinterpreted during data analysis).
71. If the sample positioning is not perfectly accurate, then the
sample edge may come to rest on top of the sample carrier ring,
preventing thermal contact between the silver block and the
sample’s bottom coverslip. Thus, one should check whether
the sample is laying flat inside the carrier ring, by nudging the
sample horizontally (lightly touching the sample using the
edge of the vacuum tweezer’s suction cup works well for this
purpose). It is also advisable at this time to move the carrier
back and forth using the BCS196 stage X- and Y-drives while
observing the sample to confirm that it can be positioned
laterally without problems (in particular, check that the carrier
ring does not slide into the space between the sample’s top and
bottom coverslip).
72. For record-keeping and quality assurance purposes, it is useful
to have a continuous recording of the complete temperature
history experienced by the sample, from the time of loading
into the cryomicroscope stage until the end of the experiment.
73. The purging process is also described in the BCS196 User
Guide, and a video tutorial can be viewed on the Linkam
Scientific Instruments website (http://www.linkam.co.uk/
video-manuals/). Purging is necessary to reduce the moisture
content within the cryomicroscope stage chamber, to prevent
condensation and frost from forming on the sample during
freezing.
74. If the LNP flowrate is increased rapidly, the sample temperature may temporarily drop (by ~1
C). If the specimen is
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Jens O. M. Karlsson
undesirable).
69. The techniques described here will yield a coverslip sandwich in
which the thickness of the liquid sample is approximately
30–50 μm. This sample thickness is appropriate for cells with
diameters in the range 10–30 μm. If necessary, the sample
thickness can be varied by altering the amount of grease or
the liquid sample volume. However, the sample thickness
should be kept as small as possible without deforming the cells.
70. It is important that the surfaces and edges of the sample be
completely dry before it is loaded into the cryomicroscope
stage. Residual liquid on the sample can cause serious problems, including contamination of the silver block surface
(which can affect temperature control), capillary adhesion of
the bottom coverslip to the silver block (which may cause the
sandwich construct to delaminate when attempting to move
the sample), and ice formation on the external surfaces of the
coverslips (which will cause the view of the sample to be
obscured, or result in confounding optical interference that
can be misinterpreted during data analysis).
71. If the sample positioning is not perfectly accurate, then the
sample edge may come to rest on top of the sample carrier ring,
preventing thermal contact between the silver block and the
sample’s bottom coverslip. Thus, one should check whether
the sample is laying flat inside the carrier ring, by nudging the
sample horizontally (lightly touching the sample using the
edge of the vacuum tweezer’s suction cup works well for this
purpose). It is also advisable at this time to move the carrier
back and forth using the BCS196 stage X- and Y-drives while
observing the sample to confirm that it can be positioned
laterally without problems (in particular, check that the carrier
ring does not slide into the space between the sample’s top and
bottom coverslip).
72. For record-keeping and quality assurance purposes, it is useful
to have a continuous recording of the complete temperature
history experienced by the sample, from the time of loading
into the cryomicroscope stage until the end of the experiment.
73. The purging process is also described in the BCS196 User
Guide, and a video tutorial can be viewed on the Linkam
Scientific Instruments website (http://www.linkam.co.uk/
video-manuals/). Purging is necessary to reduce the moisture
content within the cryomicroscope stage chamber, to prevent
condensation and frost from forming on the sample during
freezing.
74. If the LNP flowrate is increased rapidly, the sample temperature may temporarily drop (by ~1
C). If the specimen is
260
Jens O. M. Karlsson