change using video or still micrography. However, the actual ice
crystals that form inside rapidly cooled cells cannot be detected by
conventional video imaging, whereas the familiar darkening events
are due to secondary processes, which are not triggered until after
ice has already filled the cell [23]. Due to this decoupling of the cell
darkening reaction from the preceding intracellular ice formation
event (and the attendant time delay between the two phenomena),
significant errors in the estimation of intracellular ice formation
kinetics can result if darkening is used as a proxy indicator for the
crystallization of cell water [23].
The formation of ice crystals in rapidly cooled cells can be
visualized using high-speed video cryomicroscopy [23]. Ultraslow motion playback of high-speed video recordings reveals that
when ice crystals fill supercooled cells, there is no appreciable
change in cell opacity, whereas the advancing ice-liquid interface
is readily discernible in the form of a solitary wavefront that travels
through the cell interior [23]. Because the crystal growth velocity is
typically on the order ~10 μm/ms, while somatic cell diameters
tend to be on the order ~10 μm, cryomicroscopy images must be
sequentially acquired at a temporal resolution (i.e., sampling interval) no longer than 0.1–1 ms to allow accurate detection of intracellular ice formation events. Thus, to advance our understanding
of the mechanisms of intracellular ice crystallization, and to measure the true kinetics of this deleterious phase transformation process, video recordings of cryomicroscopy experiments must use
image acquisition rates in the range 10
3 –10
4 frames per second
(fps), or better. In contrast, conventional analog video recording
technology is constrained to frame rates fixed at 29.97 fps (National
Television System Committee standard) or 25 fps (Phase Alternating Line standard), yielding a temporal resolution (33 or 40 ms,
respectively) that is insufficient to capture intracellular crystallization events. With digital imaging technology, the situation is even
worse: off-the-shelf solutions that are commercially available at the
time of this writing have inferior temporal resolution and are not
suitable for detection of intracellular ice formation. For example,
turnkey systems in which digital image acquisition is integrated
with cryomicroscope temperature measurement and control
include the software products LINK-DV (Linkam Scientific Instruments, Surrey, UK) and PAX-it (Midwest Information Systems,
Villa Park, IL); in the former, the best temporal resolution for
digital video acquisition is 100 ms [24], and in the latter, the
shortest interval between successive images is 5 s [25].
Because imaging at sub-millisecond temporal resolution is
required to study intracellular ice formation processes, whereas
high-speed video cryomicroscopy systems are not yet commercially
available, this chapter presents solutions for integrating a thirdparty high-speed digital video camera with an off-the-shelf cryomicroscope stage. In addition, standard protocols for conducting a
High-Speed Video Cryomicroscopy
223
crystals that form inside rapidly cooled cells cannot be detected by
conventional video imaging, whereas the familiar darkening events
are due to secondary processes, which are not triggered until after
ice has already filled the cell [23]. Due to this decoupling of the cell
darkening reaction from the preceding intracellular ice formation
event (and the attendant time delay between the two phenomena),
significant errors in the estimation of intracellular ice formation
kinetics can result if darkening is used as a proxy indicator for the
crystallization of cell water [23].
The formation of ice crystals in rapidly cooled cells can be
visualized using high-speed video cryomicroscopy [23]. Ultraslow motion playback of high-speed video recordings reveals that
when ice crystals fill supercooled cells, there is no appreciable
change in cell opacity, whereas the advancing ice-liquid interface
is readily discernible in the form of a solitary wavefront that travels
through the cell interior [23]. Because the crystal growth velocity is
typically on the order ~10 μm/ms, while somatic cell diameters
tend to be on the order ~10 μm, cryomicroscopy images must be
sequentially acquired at a temporal resolution (i.e., sampling interval) no longer than 0.1–1 ms to allow accurate detection of intracellular ice formation events. Thus, to advance our understanding
of the mechanisms of intracellular ice crystallization, and to measure the true kinetics of this deleterious phase transformation process, video recordings of cryomicroscopy experiments must use
image acquisition rates in the range 10
3 –10
4 frames per second
(fps), or better. In contrast, conventional analog video recording
technology is constrained to frame rates fixed at 29.97 fps (National
Television System Committee standard) or 25 fps (Phase Alternating Line standard), yielding a temporal resolution (33 or 40 ms,
respectively) that is insufficient to capture intracellular crystallization events. With digital imaging technology, the situation is even
worse: off-the-shelf solutions that are commercially available at the
time of this writing have inferior temporal resolution and are not
suitable for detection of intracellular ice formation. For example,
turnkey systems in which digital image acquisition is integrated
with cryomicroscope temperature measurement and control
include the software products LINK-DV (Linkam Scientific Instruments, Surrey, UK) and PAX-it (Midwest Information Systems,
Villa Park, IL); in the former, the best temporal resolution for
digital video acquisition is 100 ms [24], and in the latter, the
shortest interval between successive images is 5 s [25].
Because imaging at sub-millisecond temporal resolution is
required to study intracellular ice formation processes, whereas
high-speed video cryomicroscopy systems are not yet commercially
available, this chapter presents solutions for integrating a thirdparty high-speed digital video camera with an off-the-shelf cryomicroscope stage. In addition, standard protocols for conducting a
High-Speed Video Cryomicroscopy
223