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

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

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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 im-aging approach which has the best of both background removal and imaging speed.However,the com-monly 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 instru-ment design and operation largely limit their throughput of data collection.Here,we propose an ap-proach 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 robust-ness 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 cross-modality 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.

Light-sheet fluorescence microscopyBackground-free fluorescence imagingDeep learningSingle cell imagingHomogeneous illuminationSingle-molecule localization microscopy

Boxin Xue、Caiwei Zhou、Yizhi Qin、Yongzheng Li、Yuao Sun、Lei Chang、Shipeng Shao、Yongliang Li、Mengling Zhang、Chaoying Sun、Renxi He、Qian Peter Su、Yujie Sun

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State Key Laboratory of Membrane Biology,Biomedical Pioneer Innovation Center (BIOPIC),School of Life Sci-ences,Peking University,Beijing 100871,China

College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China

Academy for Advanced Interdisciplinary Studies,Peking University,Beijing 100871,China

Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory),Guangzhou 510005,China

Beijing Institute of Heart Lung and Blood Vessel Disease,Beijing Anzhen Hospital,Capital Medical University,Beijing 100029,China

Department of Geriatric Dentistry,Peking University School and Hospital of Stomatology,Beijing 100081,China

School of Biomedical Engineering,Faculty of Engineering and Information Technology,University of Technology Sydney,Sydney,NSW 2007,Australia

School of Future Technology,Peking University,Beijing 100871,China

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2017YFA050530021825401

2021

生物物理学报
中国生物物理学会 中国科学院生物物理研究所

生物物理学报

影响因子：0.355
ISSN：1000-6737
年,卷(期)：2021.7(4)
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