# Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device
## 1 Introduction  
In vitro three-dimensional (3D) culture is highly important in many biological fields. The extracellular matrix (ECM) in 3D cultures provides cellular microenvironments completely different from those of 2D cultures, as illustrated by chemical and mechanical interactions with the ECM, molecular gradients, and substrate stiffness [1; 2; 3]. Thus, cell responses in 3D culture are different from those in 2D culture. Many morphological processes, including epithelial sheet folding [4; 5; 6] and the helical motion of cells [7; 8] during tissue formation, arise only when cells are surrounded by ECM. However, imaging developed 3D structures is often difficult, especially if samples are several millimeters in size. Laser microscopic systems, such as confocal microscopy [9] and two photon microscopy [10] systems, are used to obtain detailed molecular information from cultured samples, but these laser microscopic systems have poor resolution in the \(z\)-direction when samples are imaged at a broad view with small magnification lenses (4\(\times\) and 10\(\times\)). In addition, fluorescent signals are hard to detect at deep area when an objective lens with low numerical aperture (NA) is used. Sectioning of biological samples following paraffin embedding or freezing can yield better images for thick samples, but samples may deteriorate during slicing, and it is difficult to see correlations between large scale of tissue form and molecular expression. Light sheet microscopy [11; 12; 13] can overcome these problems and image tissue-sized samples, but these samples must be transferred to a special container for imaging, a process that may damage the sample. In addition, this system is quite expensive and not accessible to all researchers. Various laser microscopy systems are now commercially available, but there is no universal system for all imaging purposes. Therefore, systems or platforms, which have broad utility and do not alter users' current experimental setup, are required to enhance image quality for large-scale tissue samples.  
The method of sample setup may result in enhanced image quality. For example, a vertical confocal observation system has been found to enhance \(z\)-resolution by magnetic rotation of the sample, but these samples are limited to a single cell level cultured in 2D [14]. By contrast, we developed a 3D culture device composed of two different hydrogel types in a cubic frame, enabling transfer and rotation of a bio-sample with ECM during culturing. The device does not deteriorate cell viability comparing to the one cultured in a dish and the size of the device can be varied depending on the sample size and applications as described previously [15]. Besides, it is capable of culturing any bio-sample and is applicable to most commercial culture devices and microscopes. However, the images taken from multiple directions were hard to align spatially as 3D data and it prevented from capturing detailed 3D structure of large sample. This study describes a novel in vitro 3D culture platform that utilizes this gel cube device to image large-scale samples by achieving fine alignment of image dataset. To overcome the drawbacks of low magnification lenses, including large focal depth and low NA, samples were scanned from six sides and the imaged data were aligned precisely by self-fluorescent microstructure fabricated by photolithography. This device is applicable to the most of experimental setup while enabled clear images in the \(x\)-\(y\)-\(z\) direction to be obtained for millimeter-sized tissue samples.