Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device

Masaya Hagiwara,Tomohiro Kawahara,Rina Nobata

doi:10.3390/app8020235

Masaya Hagiwara, Tomohiro Kawahara + Show 1 more

Open Access

https://doi.org/10.3390/app8020235

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Abstract

In vitro three-dimensional (3D) culturing is considered essential in many biological fields. However, the imaging of developed 3D formations is often difficult, especially if the size of the sample is relatively large. The z-resolution of fluorescent imaging is low using low magnification lenses (4× and 10×) due to large focal depths. This paper describes 3D culture platform enabling large scale 3D imaging by fine spatial alignment of the image dataset obtained from multiple directions. A gel cube device was employed to conduct the multi-scanning and then a self-fluorescent microstructure in a cubic frame allows us spatially align image dataset within a few pixels. By synthesizing data from multiple scans, the platform enables us to visualize millimeter-sized 3D sample structure and individual cellular actin filaments at the same time. Millimeter depth imaging of a developed bronchial tree was achieved with high z-resolution. The device, which is applicable to most microscopy systems, can enhance the image quality without modifying current systems.

Highlights

In vitro three-dimensional (3D) culture is highly important in many biological fields
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 magnification lens to obtain a broader view and larger focal distance; this results in a smaller numerical aperture (NA) and a larger focal depth
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

Summary

Introduction

In vitro three-dimensional (3D) culture is highly important in many biological fields. 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× and 10×). 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. This system is quite expensive and not accessible to all researchers

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