by silver block is not usable and should be cropped out
(because imaging of obscured regions use up memory and
bandwidth). Therefore, at microscope magnifications that are
sufficiently low to allow the full area of the silver block aperture
to fit within the camera image, one should set the ROI to a
square shape with edges that do not exceed the aperture diameter. In this scenario, the upper bound on the ROI size will be
determined by the shortest dimension of the camera sensor
(conventionally the vertical dimension). For modern highspeed cameras available at the time of this writing, the maximum vertical resolution that can be used at 2000 fps (which is
the minimum recommended frame rate for imaging of intracellular ice formation) is typically in the range 800–2000 pixels,
corresponding to maximum ROI dimensions that range from
800 Â 800 pixels to 2000 Â 2000 pixels. Nonetheless, for
many high-speed video cryomicroscopy applications, a
reduced-size ROI is preferable, because keeping the ROI
small allows for longer recording times and faster frame rates.
It should be noted that if the microscope magnification is
optimally matched to the camera pixel pitch (so that a pixel
pair spans the Rayleigh resolution limit), a typical somatic cell
can be resolved in an ROI with linear dimensions on the order
of 20 pixels. Adding to this a margin to account for sample
drift, an ROI size of 128 Â 128 pixels may be suggested as a
practical minimum. At this resolution, every gigabyte of camera memory yields approximately 16–33 s of recording time at
a frame rate of 2000 fps (the exact recording time depends
primarily on the sensor bit depth, i.e., the number of distinct
gray levels that can be resolved by each pixel); in contrast,
under the same conditions, a 800 Â 800-pixel ROI will yield
a recording time of ~0.7–1.5 s/GB, and a 2000 Â 2000-pixel
ROI will only allow for recording times in the range
~0.07–0.13 s/GB.
55. As a general rule of thumb, to avoid motion blur in video
recordings of a feature that is moving at a velocity v, the camera
exposure should not exceed the image spatial resolution,
divided by v. The spatial resolution in this context is the larger
of the camera sensor resolution (i.e., pixel pitch divided by
magnification) and the Rayleigh limit (which is on the order
~1 μm for high-speed video cryomicroscopy, as a result of longworking distance lenses and the need to stop down the condenser diaphragm for improved image contrast). Thus, for a
high-speed camera sensor with pixel pitch of ~20 μm, to image
intracellular ice formation (in which the solidification front
typically advances at a velocity v % 10 μm/ms), the maximum
exposure is 400 and 200 μs for magnifications of 5Â and 10Â,
respectively; for magnifications of 20Â and greater, the camera
256
Jens O. M. Karlsson
(because imaging of obscured regions use up memory and
bandwidth). Therefore, at microscope magnifications that are
sufficiently low to allow the full area of the silver block aperture
to fit within the camera image, one should set the ROI to a
square shape with edges that do not exceed the aperture diameter. In this scenario, the upper bound on the ROI size will be
determined by the shortest dimension of the camera sensor
(conventionally the vertical dimension). For modern highspeed cameras available at the time of this writing, the maximum vertical resolution that can be used at 2000 fps (which is
the minimum recommended frame rate for imaging of intracellular ice formation) is typically in the range 800–2000 pixels,
corresponding to maximum ROI dimensions that range from
800 Â 800 pixels to 2000 Â 2000 pixels. Nonetheless, for
many high-speed video cryomicroscopy applications, a
reduced-size ROI is preferable, because keeping the ROI
small allows for longer recording times and faster frame rates.
It should be noted that if the microscope magnification is
optimally matched to the camera pixel pitch (so that a pixel
pair spans the Rayleigh resolution limit), a typical somatic cell
can be resolved in an ROI with linear dimensions on the order
of 20 pixels. Adding to this a margin to account for sample
drift, an ROI size of 128 Â 128 pixels may be suggested as a
practical minimum. At this resolution, every gigabyte of camera memory yields approximately 16–33 s of recording time at
a frame rate of 2000 fps (the exact recording time depends
primarily on the sensor bit depth, i.e., the number of distinct
gray levels that can be resolved by each pixel); in contrast,
under the same conditions, a 800 Â 800-pixel ROI will yield
a recording time of ~0.7–1.5 s/GB, and a 2000 Â 2000-pixel
ROI will only allow for recording times in the range
~0.07–0.13 s/GB.
55. As a general rule of thumb, to avoid motion blur in video
recordings of a feature that is moving at a velocity v, the camera
exposure should not exceed the image spatial resolution,
divided by v. The spatial resolution in this context is the larger
of the camera sensor resolution (i.e., pixel pitch divided by
magnification) and the Rayleigh limit (which is on the order
~1 μm for high-speed video cryomicroscopy, as a result of longworking distance lenses and the need to stop down the condenser diaphragm for improved image contrast). Thus, for a
high-speed camera sensor with pixel pitch of ~20 μm, to image
intracellular ice formation (in which the solidification front
typically advances at a velocity v % 10 μm/ms), the maximum
exposure is 400 and 200 μs for magnifications of 5Â and 10Â,
respectively; for magnifications of 20Â and greater, the camera
256
Jens O. M. Karlsson