Breakthrough in Long-term Live Subcellular Imaging: Study of Confocal Scanning Light-Field Microscopy

Research Background

Long-term live cell dynamic observation is indispensable in studying physiological pathological processes such as immune response and brain function, requiring high spatiotemporal resolution and low phototoxicity. Existing confocal microscopy techniques exclude background fluorescence through optical sectioning and improve signal-to-noise ratio (SNR), but it is difficult to strike a balance between parallel processing, resolution, and phototoxicity. Light-field microscopy, although it enhances parallel processing and has low phototoxicity, falls short in excluding background.

The “Confocal Scanning Light-Field Microscopy (CSLFM)” adopts axially extended linear confocal illumination and rolling shutter, further enhancing three-dimensional (3D) imaging quality, speed, and low phototoxicity. In the study, CSLFM achieved directional selectivity similar to two-photon microscopy, aiding in decoding neural mechanisms, and allows observing subcellular dynamics in optically challenging environments.

This paper will elaborate on the entire process of the study and the scientific and application value it brings concerning background suppression, high signal-to-noise ratio (SNR), low phototoxicity, and other aspects.

Source and Author Team

This research was conducted by scientists including Zhi Lu, Siqing Zuo, Minghui Shi, Jiaqi Fan, Jingyu Xie, Guihua Xiao, Li Yu, Jiamin Wu, and Qionghai Dai. The research team is from the Department of Automation, Institute of Brain and Cognitive Sciences, Tsinghua University, the Beijing Key Laboratory of Multidimensional and Multiscale Computational Photography, and also involves IDG/McGovern Institute for Brain Research, Zhejiang Hemu Technology, Hangzhou Zhuoxi Biological and Intelligent Research Institute, Tsinghua-Peking University Life Sciences Joint Center, Tsinghua Shenzhen International Graduate School, Shanghai Artificial Intelligence Laboratory, and Beijing Information Science and Technology National Research Center of Tsinghua University. The article was published in Nature Biotechnology.

Research Content

a) Research Process

The research includes four main procedures: research design, sample preparation, experiment execution, and data analysis. The team designed a linear confocal illumination system based on a rolling shutter and constructed both inverted and upright CSLFM systems. Experimental subjects include different species (mice, fruit flies) and tissue types. Multiple repeated experiments were carried out using the CSLFM technology, ensuring high repeatability and stability of the experiments by real-time multicolor laser illumination and signal detection.

b) Research Results

CSLFM was successfully used for subcellular structure and dynamic observation of various species, such as migratory body transmission in mouse spleen, withdrawal body formation in mouse liver, and 3D voltage imaging in fruit flies. This technology significantly improved the signal-to-noise ratio, achieving long-term, high-resolution imaging, and obtained directional selectivity of nerve responses similar to two-photon microscopy.

c) Conclusion and Significance

CSLFM provides a technical solution for long-term, high-quality three-dimensional imaging of subcellular dynamics in vivo, achieving microscopic observation with low phototoxicity and high data throughput, thereby promoting the development of biomedical research.

d) Research Highlights

Key findings of this research include the dynamic interaction between immune cells, new insights into neural coding mechanisms, and innovative CSLFM technology, demonstrating unprecedented advantages and application prospects in the field of live subcellular imaging.

Research Value and Impact

The introduction of CSLFM greatly advances interdisciplinary research in the field of life sciences, providing more accurate and in-depth observation methods for fields such as cell biology, neuroscience, and immunology, enabling researchers to study real biological processes more closely.

In addition, the author team provided detailed experimental methods, enhancing the transparency and reproducibility of the research, ensuring that this technology can be more widely applied and verified.