# Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device
## 3 Results
### Multi-Directional Imaging of a Large Branching Structure with a Low Magnification Lens  
To demonstrate the capability of the gel cube device with a fine alignment mark, normal human bronchial epithelial (NHBE) cells were cultured to develop branches [23] in the fabricated cube and actin filaments were imaged. Figure 3A shows the projected images of the developed branching structure in three planes, based on \(z\)-stack imaging data obtained from a single side. Imaging of the millimeter-sized branching structure with a 10\(\times\) magnification lens (NA 0.45) resulted in very poor resolution in the \(z\)-direction because of the large focal depth. Moreover, despite the expansion of branches, the signal was barely detected along the \(z\)-direction due to small NA. By contrast, Figure 3B shows projected image data obtained from six sides to the three planes. Following fine image alignment based on the alignment mark, the intensities of \(z\)-stack images from opposite sides (bottom and top, left and right, front and back) were averaged. These images are shown to be considerably superior to those in Figure 3A, as actin filaments in the \(y\)-\(z\), \(z\)-\(x\) and \(x\)-\(y\) planes can be clearly recognized, and the entire structure can be determined precisely. This large-scale imaging with a low magnification lens resulted in a resolution high enough to detect actin filaments in the \(z\)-direction. The poor \(z\)-resolution resulting from the large focal depth was overcome by compensation with the imaging data obtained from multiple directions. In addition, assembling projected images from six sides can clearly show an overview of the entire structure of millimeter-sized bronchial trees in any arbitrary direction (Figure 3C and Supplemental Video S1). Figure 3: Comparison of projected and section images of a developed bronchial tree in a gel cube taken from a single ordinal side and from six sides. Actin filaments were stained. The objective lens used had 10\(\times\) magnification (NA 0.45), and the scanning area was 1087 \(\times\) 1450 \(\times\) 1087 \(\upmu\)m. (**A**) Images of scanning \(z\)-stack data projected from one side to the \(x\)-\(y\), \(y\)-\(z\) and \(z\)-\(x\) planes. Most undetected signals were at the high \(z\)-portion, and resolution was very low along the \(z\)-direction. (**B**) Projection images of three datasets obtained from six scanning datasets to the \(x\)-\(y\), \(y\)-\(z\) and \(z\)-\(x\) planes. The image quality was sufficiently high to clearly show actin filaments in all planes, whereas the millimeter-sized whole branch sample was covered. (**C**) Assembled projected images from six sides. Shown is an overview of the entire structure of a millimeter-scaled bronchial tree, which can be clearly viewed from any arbitrary direction. (**D**) Section images taken from one side. In the \(x\)-\(y\) plane, the developed branch had a hollow structure. These findings were not supported by images in the \(y\)-\(z\) and \(z\)-\(x\) planes due to undetected areas on the upper sides of the branches. (**E**) Section images taken from six sides. Images in the \(x\)-\(y\), \(y\)-\(z\) and \(z\)-\(x\) planes showed that the branch had a hollow structure. Scale bar: 100 \(\upmu\)m.  
The section images obtained from one side (Figure 3D) and six sides (Figure 3E) are also shown. The image in the \(x\)-\(y\) plane of Figure 3D suggested that branches had hollow structures, whereas the images in the \(y\)-\(z\) and \(z\)-\(x\) planes provided a negative impression due to the undetected signals on the upper side of the tube. By contrast, the hollow structure was clearly imaged in all three planes (\(x\)-\(y\), \(y\)-\(z\) and \(z\)-\(x\)) of Figure 3E (Supplemental Video S2). These section images were taken at relatively large focal depth in each plane, with the sections being approximately 20-50 \(\upmu\)m thick, or only 2-4% of the view range of a 10\(\times\) objective lens.