Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2 × 2 mm² region within an imaging depth of ∼5 μm for mammalian cells in <10 min and immunofluorescence imaging at a >30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multicolor imaging, depth control, and super-resolution imaging.

中文翻译：

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2.5D 显微镜：通过体积投影进行快速、高通量成像，用于定量亚细胞分析

基于成像的单细胞分析对于研究亚细胞分辨率下生物分子的表达水平和功能至关重要。然而，它的低吞吐量阻碍了以快速有效的方式测量来自多个小区的众多细胞特征。在这里，我们报告了 2.5D 显微镜，它显着提高了荧光成像系统的吞吐量，同时保持了高分辨率和单分子灵敏度。代替顺序z扫描，体积信息通过工程发射的荧光在单次拍摄中投影到 2D 图像平面上。^{我们的方法在特定成像深度内提供了改进的成像速度和均匀的焦点响应，这使我们能够在 2 × 2 mm 2}上进行定量单分子 RNA 测量哺乳动物细胞在<10分钟内成像深度为~5μm的区域，免疫荧光成像在> 30 Hz体积帧速率下，光漂白减少。我们的显微镜还提供多色成像、深度控制和超分辨率成像的能力。