# Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device
## 4 Discussion and Conclusions  
We have achieved to develop the platform enabling fine alignment of obtained datasets by multi-scanning by employing gel cube device in order to improve the resolution in the \(z\)-direction significantly, despite using a low magnification lens. A millimeter-sized branching structure could be successfully imaged without slicing the sample or employing very expensive illumination microscopy  
Figure 4: Reconstructed 3D voxel image for the developed bronchial tree. The bronchial tree was stained for expression of actin and FGFR2. The scanned area measured 1087 \(\times\) 1450 \(\times\) 1087 \(\upmu\)m. (**A**) Image reconstructed from scanning data taken at a single side. Signals corresponding to both actin and FGFR2 could not be detected from half the developed branches. (**B**) Image reconstructed from scanning data taken from six sides. The fluorescent intensities were averaged to show the capability of the gel cube device. The entire structure could be clearly observed by staining for actin and FGFR2.  
systems. The projection image obtained from multi-direction showed the entire bronchial tree structure, without encountering focal depth problems, and section images revealed the tubular structure inside the branches. Actin filaments and FGFR2 were displayed clearly. By contrast, conventional one-sided imaging showed only half the sample with low \(z\)-resolution. The algorithm used in this study to develop multi-directional images into voxel data was a simple average of the intensity values from multiple datasets. Other sophisticated algorithms [24; 25] also have the potential to improve image quality of 3D reconstructions.  
Multi-directional imaging systems, such as light sheet fluorescent microscopy and optical projection tomography, have been already available but the obtained images in these systems are based on planar rotation of the sample, while the methodology described in this paper supplies image datasets from three axes. In addition, the platform developed in this study is compatible with most available microscopes and culture systems, and therefore does not need to modify current experimental conditions. Other technologies that enhance image quality, including super-resolution systems, tissue transparent reagents, and molecular labeling, can be applied simultaneously; however, the gel cube device can further enhance imaging data by multi-directional scanning. In addition, the production cost of the hybrid gel cube with alignment mark is surprisingly low because only widely prevailing commercial products are employed. This device has the potential to promote research required for 3D cultures, e.g., in developmental biology, drug development, and regenerative medicine.  
The following are available online at www.mdpi.com/2076-3417/8/2/235/s1, Supplemental Video S1: All-around view of assembled projected images from six sides shown in Figure 3C, Supplemental Video S2: Motion picture of Figure 3E to demonstrate spatial synchronization of the section images from multiple directions, Supplemental Video S3: All-around view of reconstructed 3D image from scanning data taken at a single side as shown in Figure 4A, Supplemental Video S4: All-around view of reconstructed 3D image from scanning data taken at six sides as shown in Figure 4B.  
This work was financially supported by JSPS KAKENHI (15H05512) and the Program to Disseminate Tenure Track System, MEXT, Japan.  
[PERSON] and [PERSON] conceived and designed the experiments; [PERSON] performed the experiments; [PERSON] and [PERSON] analyzed the data; [PERSON] and [PERSON] wrote the paper.  
The cubic culture device, which was previously published, was licensed to a company (Nippon Medical and Chemical Instruments Co. Ltd., Osaka, Japan) for commercialization. Authors declare no competing interest for the technologies in this paper.