## 4 Discussion  
The advantage of this study is that a methodology to collect real-time images at the stretching and relaxation phases was established and tested. Based on the developed method, a system was developed with the capability of taking high time resolution video clips of living cells exposed to cyclic stretching continuously. Our system, therefore, provides the possibility that even unexpected cellular mechanoresponses can be detected during experiments. To the best of our knowledge, no existing research has been developed using this technique to observe cells during the two phases. Indeed, several studies have fabricated a system to get two images during a cycle: one image before the stretching phase, and the other in the holding phase. Conventionally, the focus plate of the chamber membrane will be changed after exposure to a stretch induced by a moving indenter under the chamber. To solve this problem, [PERSON] et al. [39] developed a system with a fixed indenter underneath but a vertically moving stretch chamber. Because the indenter was fixed, the vertical position of the chamber membrane on the top of the indenter was almost constant when it was stretched. Even so, there was still a vertical shift. Therefore, they measured the volume of the maximum shift, and obtained the images before and after stretching successfully. Another effort to obtain images before and after stretching is to minimize the stretching system using microfabrication techniques, and even devices for studying the behavior of individual cells have been created. For example, [PERSON] et al. [40] have developed a micro-stretching device with a width of approximately 250 \(\upmu\)m. Indeed, miniaturization of a stretching device may minimize the volume of vertical shift. However, even though the device was fabricated at the micro level, there were cases where the targets moved out of the focal plane, which may depend on the magnification of the lens or the structure of the micro-device [41; 42]. Further, the quality of imaging was always dependent on the thickness of the chamber membrane, because stretch-induced thickness change was also involved in a target position change. Therefore, a theory other than system minimization to compensate for the shift is necessary, a theme which was tried by this study. This study did not investigate the effectiveness of the methodology on real-time imaging when the chamber was exposed to a larger/smaller strain or stretching with a different type of stretch waveform (e.g., sinusoidal waveforms) other than trapezoidal ones. Indeed, if the stretch waveform was different from the waveforms used in this study, the corresponding waveforms of the vertical shift should be specially designed. One method is that researchers directly measure the displacement waveforms of the chamber membrane when exposed to cyclic stretching before experiments involved in cells, and then figure out the mathematical model of the stretching or relaxation phases, linear or non-linear ones, by curve fitting. However, a trapezoidal waveform may be the most practical one for vertical compensation of a miniaturized chamber exposed to types of stretching waveforms, and the reasons are as follows. If the vertical shift is very small (several \(\upmu\)m to 10-20 \(\upmu\)m), the averaged vertical shift compensated by the motorized stage at every millisecond should be very small during lengthening/relaxation. Because the averaged vertical shift for each millisecond is 0-2 \(\upmu\)m of a lengthening/relaxation phase in this study, the shift can be compensated by sending one or two pulses (positioning accuracy of stage 3: 1 \(\upmu\)m/pulse, and maximum drive speed of 2 \(\upmu\)m/ms).