Imaging Subcellular Dynamics with Fast and Light-Efficient Volumetrically Parallelized Microscopy.
Imaging Subcellular Dynamics with Fast and Light-Efficient Volumetrically Parallelized Microscopy.
复制标题
DOI：
10.1364/optica.4.000263
复制
发表时间：
2017-02-20
期刊：
影响因子：
10.4
通讯作者：
Fiolka R
中科院分区：
文献类型：
--
作者：
Dean KM;Roudot P;Welf ES;Pohlkamp T;Garrelts G;Herz J;Fiolka R
在荧光显微镜中，连续采集2D图像以形成3D体积限制了最大成像速度。这在以光片荧光显微镜形式对贴壁细胞成像时尤其明显，因为它们的细长形态需要每个图像体积约200个图像平面。在这里，通过用三个光片照射标本，每个光片独立检测，我们提出了一种光效率高，无串扰，体积平行化的3D显微镜技术，该技术针对贴壁细胞的高速（高达14 Hz）亚细胞（300 nm横向，600 nm轴向分辨率）成像进行了优化。我们展示了细胞内过程的3D成像，包括单细胞迁移和集体伤口愈合的细胞骨架动力学，分别为1500和1000个时间点。此外，我们捕捉快速的生物过程，包括运输速度超过每秒10微米的早期内体和初级神经元中的钙信号。
In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells. We demonstrate 3D imaging of intracellular processes, including cytoskeletal dynamics in single cell migration and collective wound healing for 1500 and 1000 time points, respectively. Further, we capture rapid biological processes, including trafficking of early endosomes with velocities exceeding 10 microns per second and calcium signaling in primary neurons.