Tips to improve sarcomere measurements
The quality of the recording of sarcomere
length depends on several parameters which can be optimized to improve the
results.
Example
of a calculation of resolution
Nota bene: According to Nyquist-Shannon theorem, the pixel size has to allow at least
4 pixels per sarcomere at the maximum of the contraction i.e. the shortest
distance between sarcomeres. In our example, for a sarcomere length of 1.5 µm,
there should be less than 103 cycles in the window size (512 pixels) i.e. ~5
pixels per sarcomere.
· Temporal resolution: set by the video camera frame rate.
The video camera frame rate (FR) can be
enhanced by selecting a Region Of Interest (ROI) and
using the binning mode. Using ROI increases the FR by reducing the used size of
the camera sensor. Binning mode increases the FR and the light sensitivity but
decreases the spatial resolution.
Here an IDS UI-1220LE-M-GL digital video camera
was used with ROI and binning modes for a final resolution of
376 x 24 pixels allowing a FR of 1000 Hz. This FR is rapid enough to
record sarcomere length in contracting rat ventricular cardiomyocytes.
Analogical video acquisition with a frame
grabber is generally limited to 25 – 30 Hz FR or requires very specific
material. Thus, it is better to use digital video cameras with FR which can
reach 250 Hz with binning and ROI modes. These cameras are often not
expensive (~ 300 $).
· Light and contrast: the more contrast between isotropic
and anisotropic bands, the higher is the peak in the FFT spectrum. This peak
should be as narrow and high as possible. Be sure to obtain a single peak.
· Focal plane: depth of field should be as small
as possible to improve the signal.
· Analysis axis: the analysis axis drawn along the
longitudinal axis of the cardiomyocyte has to be as perpendicular as possible
to sarcomeres. The quality of the FFT spectrum is greater when the length of
the axis is equal to 
.
is
recommended.