清华大学戴琼海团队的最新研究提出了用共聚焦扫描光场显微镜进行长期活体亚细胞成像。相关论文于2024年5月27日发表于国际顶尖学术期刊《自然—生物技术》杂志上。

据介绍，生物体内亚细胞动力学的长期观察受到来自组织散射或密集标记的背景荧光的限制。现有的共聚焦方法在并行化、分辨率和光毒性之间面临着不可避免的权衡。

该课题组研究人员提出了共聚焦扫描光场显微镜(csLFM)，它集成了轴向拉长线共聚焦照明与扫描光场显微镜(sLFM)的滚动快门。csLFM能够以接近衍射极限的分辨率实现高保真、高速、三维(3D)成像，同时具有光学切片和低光毒性的特性。通过同时进行三维激发和检测，可以将激发强度降低到1 mW mm^-2、比sLFM高15倍的信噪比。

该研究组在不同物种的光学挑战性环境中，对25000个时间框架内的亚细胞动力学进行了成像，例如同种动物脾脏的迁移体递送，同种动物肝脏的收缩体产生以及果蝇的3D电压成像。

此外，csLFM有助于高保真、大规模的神经记录，减少串扰，导致对视觉刺激的高度定向选择性，类似于双光子显微镜，这有助于理解神经编码机制。

附：英文原文

Title: Long-term intravital subcellular imaging with confocal scanning light-field microscopy

Author: Lu, Zhi, Zuo, Siqing, Shi, Minghui, Fan, Jiaqi, Xie, Jingyu, Xiao, Guihua, Yu, Li, Wu, Jiamin, Dai, Qionghai

Issue&Volume: 2024-05-27

Abstract: Long-term observation of subcellular dynamics in living organisms is limited by background fluorescence originating from tissue scattering or dense labeling. Existing confocal approaches face an inevitable tradeoff among parallelization, resolution and phototoxicity. Here we present confocal scanning light-field microscopy (csLFM), which integrates axially elongated line-confocal illumination with the rolling shutter in scanning light-field microscopy (sLFM). csLFM enables high-fidelity, high-speed, three-dimensional (3D) imaging at near-diffraction-limit resolution with both optical sectioning and low phototoxicity. By simultaneous 3D excitation and detection, the excitation intensity can be reduced below 1mWmm2, with 15-fold higher signal-to-background ratio over sLFM. We imaged subcellular dynamics over 25,000 timeframes in optically challenging environments in different species, such as migrasome delivery in mouse spleen, retractosome generation in mouse liver and 3D voltage imaging in Drosophila. Moreover, csLFM facilitates high-fidelity, large-scale neural recording with reduced crosstalk, leading to high orientation selectivity to visual stimuli, similar to two-photon microscopy, which aids understanding of neural coding mechanisms.

DOI: 10.1038/s41587-024-02249-5

Source: https://www.nature.com/articles/s41587-024-02249-5