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 μm³ 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.

微型荧光显微镜是系统生物学的标准工具。然而，宽视场微型显微镜仅捕获 2D 信息，而启用 3D 功能的修改会增加尺寸和重量，并且在狭窄深度范围之外的分辨率较差。在这里，我们通过在物镜孔径光阑处用优化的多焦点相位掩模替换传统 2D 微型显微镜的管透镜来实现 3D 功能。将相位掩模板放置在孔径光阑处可以显着减小器件的尺寸，并且改变焦距可以在较宽的深度范围内实现均匀的分辨率。相位掩模将 3D 荧光强度编码为单个 2D 测量结果，并通过解决稀疏约束逆问题来恢复 3D 体积。我们提供设计和制造相位掩模的方法以及考虑微型物镜中场变像差的有效正演模型。我们展示了一个高 17 毫米、重 2.5 克的原型，以每秒 40 个体积的速度在 900 × 700 × 390 μm ³体积的大部分区域实现 2.76 μm 横向分辨率和 15 μm 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进行了实验验证。与现有的微型单次体积捕获实现相比，我们的系统更小、更轻，并且在 10 倍大的可用深度范围内实现了 2 倍以上的横向和轴向分辨率。我们的显微镜设计为需要紧凑平台的应用提供单次 3D 成像，例如自由移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 3D 运动研究。