National Taiwan University, Institute of Medical Device and Imaging, Taipei, Taiwan.
National Tsing Hua University, Department of Power Mechanical Engineering, Hsinchu, Taiwan.
J Biomed Opt. 2020 Nov;25(12). doi: 10.1117/1.JBO.25.12.123705.
Two-photon (2P) fluorescence imaging can provide background-free high-contrast images from the scattering tissues. However, obtaining a multiplane image is not straightforward. We present a two-photon volume holographic imaging (2P-VHI) system for multiplane imaging.
Our goal was to design and implement a 2P-VHI system that can provide the high-contrast optically sectioned images at multiple planes.
A 2P-VHI system is presented that incorporates angularly multiplexed volume holographic gratings and a femtosecond laser source for fluorescence excitation for multiplane imaging. A volume hologram with multiplexed gratings provides multifocal observation, whereas nonlinear excitation using a femtosecond laser helps in significantly enhancing both depth resolution and contrast of images.
Standard fluorescent beads are used to demonstrate the imaging performance of the 2P-VHI system. Two-depth resolved optical-sectioning images of fluorescently labeled thick mice intestine samples were obtained. In addition, the optical sectioning capability of our system is measured and compared with that of a conventional VHI system.
Results demonstrated that 2P excitation in VHI systems provided the optical sectioning ability that helps in reducing background noise in the images. Integration of nonlinear fluorescence excitation in the VHI provides some unique advantages to the system and has potential to design multidepth optical sectioned spatial-spectral imaging systems.
双光子（2P）荧光成像是一种可以从散射组织中提供无背景高对比度图像的技术。然而，获得多层面图像并不简单。我们提出了一种用于多层面成像的双光子体全息成像（2P-VHI）系统。
我们的目标是设计和实现一种能够在多个平面上提供高对比度光学切片图像的 2P-VHI 系统。
提出了一种 2P-VHI 系统，该系统结合了角复用体全息光栅和飞秒激光源用于荧光激发，用于多层面成像。具有复用光栅的体全息图提供多焦点观察，而使用飞秒激光进行非线性激发有助于显著提高图像的深度分辨率和对比度。
使用标准荧光珠来演示 2P-VHI 系统的成像性能。获得了荧光标记的厚老鼠肠样本的两个深度分辨光学切片图像。此外，还测量并比较了我们系统的光学切片能力与传统 VHI 系统的能力。
结果表明，VHI 系统中的 2P 激发提供了光学切片能力，有助于减少图像中的背景噪声。将非线性荧光激发集成到 VHI 中为系统提供了一些独特的优势，并有可能设计出多深度光学切片空间光谱成像系统。
