## 3 Results
### Cell Observation with a High Time Resolution  
Finally, we applied cyclic stretching to the cells by using this system. The system was designed to be compact, which can be fit to the stages of conventional microscopes. In this study, an Olympus CK2 (Olympus) or the EVOS digital inverted microscope (Advanced Microscopy Group) were applied. Olympus CK2 was mainly used to investigate the effectiveness of the designed compensation strategy, and the EVOS digital microscope was used for the collection of online images of cells with transmitted light (phase contrast). Cell culture: The MC3T3-E1 cell line (American Type Culture Collection, Manassas, VA, USA) from mouse was used, which was first cultured in Minimum Essential Medium (MEM) \(\alpha\) (Sigma-Aldrich, Saint Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, CA, USA) in a flask, and then were detached with 0.05% trypsin-EDTA and seeded at a concentration of \(2\times 10^{4}\) cells/mL on fibronectin-coated membranes in a stretch chamber, and maintained at 37 \({}^{\circ}\)C in a humidified atmosphere for 24 h.  
Cyclic stretch exposure and image acquisition: Cells on an elastic membrane under static conditions exhibit cobble-stone-like shapes with random orientation. We applied 10% cyclic stretch (stage 1 moved for 2 mm in 1 sec) to cells for 90 min. In the experiment with living cells, we added a semi-closed stage incubator to maintain the cell culture temperature (37 \({}^{\circ}\)C). Using a lens with a magnitude ratio 20\(\times\) or 40\(\times\), the phase contrast images of cells in the FOV was monitored over a 90-min period by capturing 30 images every 1 s.  
An online cell movie was obtained using a lens with a magnification of 20\(\times\), as shown in Figure 11 and Video S2. The movie shows that targets (cells for investigation) were observed clearly during a period of approximately 90 min. Figure 9: An artificial marker was used in the basic experiments to test the effectiveness of compensation. The diameter of the marker was approximate 50 \(\upmu\)m. Images at the beginning, middle, and end of a lengthening phase are shown from the left to right. (**a**) Chamber stretching by stage 1 only (conventional). (**b**) Chamber stretching by stage 1 with compensation in the horizontal direction by stage 2. (**c**) Stretching by stage 1 and compensation in horizontal (stage 2) and vertical direction (stage 3). Figure 10: Stretch waveform of stage 1 and calculated contrast for each condition. In this experiment, maximum \(\delta x\) and \(\delta z\) were 1.25 mm and 0.11 mm, respectively. (**a**) The chamber stretching waveform by stage 1. (**b**) Contrast values of point 3 during the stretching/relaxation process without compensation (corresponding to Figure 9a). (**c**) Contrast values with the compensation in the horizontal direction (stage 2) (corresponding to Figure 9b). (**d**) Contrast values with the compensation by stage 2 and 3 (corresponding to Figure 9c). By using this system, conventional cell analysis is certainly possible (Figure 11), and further, detailed cellular responses within several seconds can be only figured out from the movie using the developed system with a high time resolution in this study (Figure 12). As shown in Figure 11a, we can roughly analyze the morphological change of a single cell exposed to cyclic stretching from the cell movie at the time point of 0, 45, and 90 min. After exposure to a cyclic stretch for 90 min, the cell enclosed by the broken line in Figure 11a changed its shape and orientation, a phenomenon also observed using the conventional stretching systems, although the results in most of the previous studies were those analyzed from different samples with cell fixation [49; 50]. Figure 11: Cell morphological response after cyclic stretch exposure using the developed system. Images were captured from a real-time movie. (**a**) The cell enclosed by the dashed line was selected as the target, and images at the beginning, middle, and end of experiments are shown here.