微型3D显微镜:优化的单次微型3D荧光显微镜

Miniscope3D: optimized single-shot miniature 3D fluorescence microscopy.

作者信息

Yanny Kyrollos, Antipa Nick, Liberti William, Dehaeck Sam, Monakhova Kristina, Liu Fanglin Linda, Shen Konlin, Ng Ren, Waller Laura

机构信息

UCB/UCSF Joint Graduate Program in Bioengineering, University of California, Berkeley, CA 94720 USA.

Department of Electrical Engineering & Computer Sciences, University of California, Berkeley, CA 94720 USA.

出版信息

Light Sci Appl. 2020 Oct 2;9:171. doi: 10.1038/s41377-020-00403-7. eCollection 2020.

Abstract

Miniature fluorescence microscopes are a standard tool in systems biology. However, widefield miniature microscopes capture only 2D information, and modifications that enable 3D capabilities increase the size and weight and have poor resolution outside a narrow depth range. Here, we achieve the 3D capability by replacing the tube lens of a conventional 2D Miniscope with an optimized multifocal phase mask at the objective's aperture stop. Placing the phase mask at the aperture stop significantly reduces the size of the device, and varying the focal lengths enables a uniform resolution across a wide depth range. The phase mask encodes the 3D fluorescence intensity into a single 2D measurement, and the 3D volume is recovered by solving a sparsity-constrained inverse problem. We provide methods for designing and fabricating the phase mask and an efficient forward model that accounts for the field-varying aberrations in miniature objectives. We demonstrate a prototype that is 17 mm tall and weighs 2.5 grams, achieving 2.76 μm lateral, and 15 μm axial resolution across most of the 900 × 700 × 390 μm volume at 40 volumes per second. The performance is validated experimentally on resolution targets, dynamic biological samples, and mouse brain tissue. Compared with existing miniature single-shot volume-capture implementations, our system is smaller and lighter and achieves a more than 2× better lateral and axial resolution throughout a 10× larger usable depth range. Our microscope design provides single-shot 3D imaging for applications where a compact platform matters, such as volumetric neural imaging in freely moving animals and 3D motion studies of dynamic samples in incubators and lab-on-a-chip devices.

摘要

微型荧光显微镜是系统生物学中的标准工具。然而,宽视场微型显微镜仅能捕捉二维信息,而实现三维功能的改进会增加尺寸和重量,并且在狭窄深度范围之外分辨率较差。在此,我们通过在物镜的孔径光阑处用优化的多焦点相位掩膜替换传统二维微型显微镜的镜筒透镜来实现三维功能。将相位掩膜置于孔径光阑处可显著减小设备尺寸,改变焦距可在较宽深度范围内实现均匀分辨率。相位掩膜将三维荧光强度编码为单个二维测量值,通过求解稀疏约束反问题来恢复三维体积。我们提供了设计和制造相位掩膜的方法以及一个考虑微型物镜中场变化像差的高效正向模型。我们展示了一个原型,其高度为17毫米,重量为2.5克,在每秒40帧的情况下,在900×700×390微米的大部分体积内实现了2.76微米的横向分辨率和15微米的轴向分辨率。该性能在分辨率目标、动态生物样本和小鼠脑组织上通过实验得到了验证。与现有的微型单次体积捕获实现方式相比,我们的系统更小、更轻,并且在10倍更大的可用深度范围内实现了超过2倍的更好横向和轴向分辨率。我们的显微镜设计为紧凑平台至关重要的应用提供了单次三维成像,例如自由活动动物的体积神经成像以及培养箱和芯片实验室设备中动态样本的三维运动研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9daa/7532148/c01d6ab7212a/41377_2020_403_Fig1_HTML.jpg

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