PN-ImTLSM facilitates high-throughput low background single-molecule localization microscopy deep in the cell

查看全文 作　　者：Boxin [1,4]Xue;Caiwei [2]Zhou;[1]YizhiQin;Yongzheng [1,2]Li;Yuao [1]Sun;Lei [1,5]Chang;Shipeng [1,6]Shao;Yongliang [7]Li;Mengling [1]Zhang;Chaoying [1]Sun;Renxi [1]He;Qian Peter [1,8]Su;Yujie [1,3]Sun 机构地区：[1]State Key Laboratory of Membrane Biology,Biomedical Pioneer Innovation Center(BIOPIC),School of Life Sciences,Peking University,Beijing 100871,China;[2]Academy for Advanced Interdisciplinary Studies,Peking University,Beijing 100871,China;[3]School of Future Technology,Peking University,Beijing 100871,China;[4]College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China;[5]Bioland Laboratory(Guangzhou Regenerative Medicine and Health Guangdong Laboratory),Guangzhou 510005,China;[6]Beijing Institute of Heart Lung and Blood Vessel Disease,Beijing Anzhen Hospital,Capital Medical University,Beijing 100029,China;[7]Department of Geriatric Dentistry,Peking University School and Hospital of Stomatology,Beijing 100081,China;[8]School of Biomedical Engineering,Faculty of Engineering and Information Technology,University of Technology Sydney,Sydney,NSW 2007,Australia 出　　处：《Biophysics Reports》索引2021年第7卷第4期,共13页 基　　金：We thank Prof.Wei Guo of the University of Pennsylvania for providing the Hela-S3 cell line(PubMed ID:5733811);This work is supported by grants from the National Key R&D Program of China for Y.Sun(2017YFA0505300);the National Science Foundation of China for Y.Sun(21825401)。 摘　　要：When imaging the nucleus structure of a cell,the out-of-focus fluorescence acts as background and hinders the detection of weak signals.Light-sheet fluorescence microscopy(LSFM)is a wide-field imaging approach which has the best of both background removal and imaging speed.However,the commonly adopted orthogonal excitation/detection scheme is hard to be applied to single-cell imaging due to steric hindrance.For LSFMs capable of high spatiotemporal single-cell imaging,the complex instrument design and operation largely limit their throughput of data collection.Here,we propose an approach for high-throughput background-free fluorescence imaging of single cells facilitated by the Im-mersion Tilted Light Sheet Microscopy(ImTLSM).ImTLSM is based on a light-sheet projected off the optical axis of a water immersion objective.With the illumination objective and the detection objective placed opposingly,ImTLSM can rapidly patrol and optically section multiple individual cells while maintaining single-molecule detection sensitivity and resolution.Further,the simplicity and robustness of ImTLSM in operation and maintenance enables high-throughput image collection to establish background removal datasets for deep learning.Using a deep learning model to train the mapping from epi-illumination images to ImTLSM illumination images,namely PN-ImTLSM,we demonstrated crossmodality fluorescence imaging,transforming the epi-illumination image to approach the background removal performance obtained with ImTLSM.We demonstrated that PN-ImTLSM can be generalized to large-field homogeneous illumination imaging,thereby further improving the imaging throughput.In addition,compared to commonly used background removal methods,PN-ImTLSM showed much better performance for areas where the background intensity changes sharply in space,facilitating high-density single-molecule localization microscopy.In summary,PN-ImTLSM paves the way for background-free fluorescence imaging on ordinary inverted microscopes. 关 键 词：Light-sheet fluorescence microscopy Background-free fluorescence imaging Deep learning Single cell imaging Homogeneous illumination Single-molecule localization microscopy