# Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device
## 3 Results  
### Large-Scale 3D Imaging of Tissue Sample with a Low Magnification Lens  
Laser microscopy systems are widely used to visualize specific molecular distribution, 3D outer structure, inner structure, and cellular characteristics (e.g., cell type, differentiation, and polarity). It frequently happens that undesired noise deteriorates the laser illumination imaging, and many studies of measurement systems [16; 17], image algorithms [18; 19], and reagent for sample preparation [20; 21] have been improved to enhance image quality. However, achieving large-scale (millimeters) high-resolution 3D imaging remains difficult, due to the trade-off between the viewing range and NA of an objective lens. Imaging of a large-scale sample like tissue, requires a low magnification lens to obtain a broader view and larger focal distance; however, this results in a smaller NA and a larger focal depth. For example, use of a 10\(\times\) magnification lens with NA 0.45 results in a theoretical focal depth of approximately 80 \(\upmu\)m, making it impossible to obtain detailed information in the \(z\)-direction. Nevertheless, \(x\)-\(y\) resolution is relatively high (1-3 \(\upmu\)m) even when using a low magnification lens (Figure 1A). Figure 1B shows measurement errors in image dimension and shape measured in \(y\)-\(z\) and \(z\)-\(x\) planes for a 10 \(\upmu\)m bead viewed by confocal microscopy. A Carl Zeiss LSM-700 confocal microscopy system was used, along with 10\(\times\) (NA 0.45), 20\(\times\) (NA 0.8) and 40\(\times\) (NA 0.95) objective lenses. As the magnification of the objective lens decreased, the measurement error of dimension and shape increased, with a 300% measurement error generated when a 10\(\times\) lens was used due to the large focal depth. By contrast, dimension and shape accuracy were relatively high in the \(x\)-\(y\) plane, even when using a low magnification lens, because there was no focal depth problem and the measurement resolution depended on the viewing range and charge-coupled device (CCD) camera resolution. By rotating  
Figure 1: Large-scale tissue imaging concept. (**A**) 3D image taken by a confocal microscope with a 40\(\times\) objective lens. The projected image of microbeads 10 \(\upmu\)m in diameter to the \(y\)-\(z\) and \(z\)-\(x\) planes was elongated due to the focal depth. (**B**) Quantitative evaluation of the relationship between magnification of objective lens and measurement errors in dimension and shape. Using a 10\(\times\) magnification lens, the measurement error for both dimension and shape was 300% in both the \(y\)-\(z\) and \(z\)-\(x\) planes, but was considerably smaller in the \(x\)-\(y\) plane. (**C**) Conceptual diagram showing a method to improve resolution in the \(z\)-direction. Deep focal depth with a low magnification lens results in a deterioration of spatial resolution. However, sample rotation after \(z\)-stack imaging could convert poor \(z\)-resolution to rich \(x\)-\(y\) resolution. (**D**) Representative image of a gel cube device. The agarose walls were sufficiently rigid to maintain extracellular matrix (ECM) and a bio-sample inside, while being sufficiently permeable to allow high molecular weight nutrients to pass into the device from the culture medium. Thus, a bio-sample can be cultured by immersing the device in culture medium and by picking up and rotating the cubic device containing ECM during culturing. (**E**) Conceptual image of large-scale imaging with a low magnification lens by the gel cube device. Rotating the sample by 90 degrees allowed multiple scanning in six directions, resulting in high-resolution imaging of millimeter-sized samples. the sample 90 degrees and obtaining \(z\)-stack images from multiple directions, it would be possible to compensate for the poor \(z\)-resolution with the \(x\)-\(y\) resolution (Figure 1C). The hybrid gel cube device (Figure 1D) allows sample rotation during 3D culturing because of its permeability to nutrients and the durability of the agarose material [15].