Department of Automation, Tsinghua University, Beijing, China.
Institute for Brain and Cognitive Sciences, Tsinghua University, Beijing, China.
Nat Biomed Eng. 2024 Jun;8(6):740-753. doi: 10.1038/s41551-023-01155-6. Epub 2023 Dec 6.
Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces.
荧光显微镜允许在哺乳动物的脑区中进行高通量的细胞活性成像。然而,由于在图像分辨率、速度、视野和景深方面存在权衡,捕捉弯曲皮质表面上的快速细胞动力学是具有挑战性的。在这里,我们报告了一种用于宽场荧光成像的技术,该技术利用选择性照明,并通过具有不同厚度的旋转盘来整合不同深度的焦点区域,从而以视频速率对以前重建的厘米级任意形状表面进行成像,其分辨率达到微米级,景深达到毫米级。通过在每秒可获取 16 亿像素、每秒可解析 168 亿体素的显微镜中实现该技术,我们在活体小鼠的弯曲皮质表面上实时记录了神经活动和中性粒细胞的轨迹。该技术可以集成到许多显微镜和宏观显微镜中,无论是反射模式还是荧光模式,都可以用于研究任意形状表面上的多尺度细胞相互作用。
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