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Cell Stretcher CS-10 Series (continued) Motion Compensation:
How does the motion A B C Figure 2
A: To demonstrate the displacement without motion compensation a membrane was stained with ink and a region 3mm away from the center of the membrane chamber was observed during stretch. A stretch of 6% was already moving most of the original area of interest out of the field of view.
B: With the motion compensation activated, the region of interest remains in the field of view of the microscope.
C: Measuring the relative displacement of 5 points on the membrane (all at a distance from the center between 2mm–4mm) we can show that even a stretch of 50% leads to only minor displacement with motion compensation. The mean relative displacements and the standard errors are shown.
compensation work? The motion compensation is not based on a feedback mechanism that recalculates the position from new image data but it is entirely based on the distance of the area of interest from the center of the membrane. To keep the specimen in the field of view of the microscope during stretch or compression of the cells the SC-device simply has to “know” the position of the membrane. This is achieved by calibrating the device once before mounting the membrane chamber onto the device and using only the software of the SC-device afterwards when the membran chamber has to be moved along the axis parallel to the stretch/compression. Our uni-directional stretch device produces a homogeneous strain field regardless of the distance to the holders where the membrane is clamped. Thus the displacement of any point is defined by a simple linear correlation that depends on the distance between the observed point from the center of the membrane. A point at the very center of the membrane is not moved out of the optical axis whereas the displacement becomes larger with increasing distance from the center. Using a simple algorithm it is possible to predict this displacement and to counteract it. The system is designed in a way that both motors — the SC-motor and the compensation motor — are operated simultaneously so that there is virtually no delay between cell stretch/compression and compensation.
C Figure 3. Image acquisition of ATII cells subject to one fast single stretch with motion compensation applied.
A: Cells were stretched with a simple protocol (20% stretch within 1s) and continuously imaged for 53.6s. The blue part of the time line illustrate the time where images were not in focus. B: Four images of the sequence (numbers are shown in A) show ratiometric images of the Ca2+
images (from low to high Ca2+ dye Fura-2. The false color
concentrations: blue-red-yellow-white) were calculated from 2 consecutive images acquired with different excitation wavelengths. Images were acquired with a 40x oil immersion objective. The position of the region was ca. 1mm off the center of the membrane parallel as well as perpendicular to the stretch axis. The vertical shift of the image between panels 1 and 2 is caused by the displacement perpendicular to the stretch axis.
C: The Ca2+ different Ca2+
signal was quantified and the time course is shown. It demonstrates that the four cells indicated show a slightly response.