Institute of Medical Device and Imaging, National Taiwan University, Taipei, Taiwan.
School of Medicine, National Taiwan University, Taipei, Taiwan.
J Biophotonics. 2018 Nov;11(11):e201800010. doi: 10.1002/jbio.201800010. Epub 2018 Jul 9.
Optical sectioning endoscopy such as confocal endoscopy offers capabilities to obtain three-dimensional (3D) information from various biological samples by discriminating between the desired in-focus signals and out-of-focus background. However, in general confocal images are formed through point-by-point scanning and the scanning time is proportional to the 3D space-bandwidth product. Recently, structured illumination endoscopy has been utilized for optically sectioned wide-field imaging, but it still needs axial scanning to acquire images from different depths of focal plane. Here, we report wide-field, multiplane, optical sectioning endoscopic imaging, incorporating 3D active speckle-based illumination and multiplexed volume holographic gratings, to simultaneously obtain images of fluorescently labeled tissue structures from different depths, without the need of scanning. We present the design, and implementation, as well as experimental data, demonstrating this endoscopic system's ability to obtain optically sectioned multiplane fluorescent images of tissue samples, with cellular level resolution in wide-field fashion, and no need for mechanical or optical axial scanning.(A) Schematic drawing of the SIHN endoscopy to simultaneously acquire multiplane images from different depths. (B) Uniform, and (C) SIHN illuminated images of standard fluorescence beads (25 μm in diameter) for the two axial planes. (D) Intensity profile on fluorescently labeled signal (ie, in-focus) and background (ie, out-of-focus) of microspheres.
光学切片内镜，如共聚焦内镜，通过区分所需的聚焦信号和离焦背景，提供了从各种生物样本中获取三维（3D）信息的能力。然而，一般来说，共聚焦图像是通过逐点扫描形成的，扫描时间与 3D 空间带宽积成正比。最近，结构光照明显微镜已被用于光学切片宽场成像，但仍需要轴向扫描才能从不同焦平面深度获取图像。在这里，我们报告了一种宽场、多平面、光学切片内镜成像技术，结合了基于 3D 主动散斑的照明和复用体全息光栅，能够同时从不同深度获得荧光标记组织结构的图像，而无需扫描。我们介绍了该内镜系统的设计、实现以及实验数据，证明了该系统能够以宽场方式获得具有细胞级分辨率的组织样本的光学切片多平面荧光图像，而无需机械或光学轴向扫描。(A) 用于同时从不同深度获取多平面图像的 SIHN 内镜的原理图。(B) 标准荧光珠（直径 25μm）的均匀和（C）SIHN 照明图像，用于两个轴向平面。(D) 微球上荧光标记信号（即聚焦）和背景（即离焦）的强度分布。
