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Comments | Xue Tian (Editor in charge of University of Science and Technology of China) | Xi Optical microscope is an important tool for biomedical research at sub-micron resolution.
The fine structure of biological tissues is complex and diverse, and how to use optical methods to observe them comprehensively and accurately in three-dimensional space is a recognized problem.
Neurons with complex shapes are the basic functional units of the brain, and how to obtain their complete structure poses a great challenge to the existing technology.
The diameter of the fluorescently labeled neuron cell body is about 10-20 microns, and the diameter of the axons and numerous branch fibers extending from the cell body is only 0.
2-0.
5 microns, and most of them project to different brain regions of the whole brain.
The brightness of the cell body and fiber differs by 2-3 orders of magnitude, and the spatial distribution is often intertwined.
To detect the weak fluorescent signal on axons under the interference of surrounding cells is like observing small stars around the bright sun.
For such situations, traditional optical tomography methods are difficult to achieve.
On March 1, 2021, the team of Academician Qingming Luo of the Biomedical Photonics Function Laboratory of Wuhan Optoelectronics National Research Center, Huazhong University of Science and Technology published an article High-definition imaging using line-illumination modulation microscopy in Nature Methods and developed line illumination Modulation microscopy and high-definition imaging are realized.
Luo Qingming's team proposed a new method of high-definition, high-throughput optical tomography—Line Illumination Modulated Optical Tomography (Line Illumination Microscopy, LiMo).
They cleverly used the Gaussian distribution of the linear illumination light intensity as a natural illumination intensity modulation mode.
With different illumination intensities, only the signal on the focusing surface produces corresponding modulation, while the background signal outside the focusing is not modulated.Therefore, the multi-line detection method can record these signals modulated by different intensities at one time, and only the simplest one-step linear calculation is needed to remove the same out-of-focus background signal and obtain a clear focal plane optical tomographic image.
Compared with classical methods such as laser spot confocal scanning microscopy, two-photon excitation fluorescence microscopy, laser line confocal scanning microscopy and structured light optical tomography, which are widely used in biomedicine, it is proved by theoretical derivation The background signal of LiMo has a faster attenuation coefficient.
This conclusion is also proved by experimental tests.
The signal-to-background ratio of the LiMo method is 1-2 orders of magnitude higher than that of the above-mentioned traditional methods.
This method only needs a simple multi-line detection line illumination light path, overcomes the inherent defects of residual modulation artifacts in traditional structured light illumination imaging, and does not require multiple imaging to obtain the required data, and has line scanning for a large range of samples The advantage of high imaging throughput solves the problem that traditional fluorescence microscopy optical tomography imaging methods cannot take into account high resolution, high throughput and high definition at the same time.
It can be said that the method, whether it is an optical path or an algorithm, fully reflects the beauty of simplicity of technology.
Fig.
1 Schematic diagram of LiMo microscopic imaging principle and performance test results Based on this, Qingming Luo’s team further developed the high-definition fluorescent micro-optical sectioning tomography (HD-fMOST), which transformed the whole brain from optical imaging.
The high resolution is raised to the new standard of high definition.
In recent years, whole-brain optical imaging has brought unprecedented rich details to biomedical research, but it has also produced huge amounts of data difficulties.
In order to solve this problem, researchers mainly focus on the algorithm field to find solutions.
Luo Qingming's team pointed out in a unique way that the fundamental strategy for solving big data problems should be to improve data quality from the source, thereby improving the efficiency of subsequent data processing and analysis.
They used HD-fMOST to perform three-dimensional high-definition two-color imaging of the mouse brain with sparsely labeled neurons, and acquired 12,000 coronal images and cell structure information within 5 days with a resolution of 0.
3 × 0.
3 × 1 micron voxel.
At present, it is the fastest technology for realizing whole-brain optical imaging with similar voxel resolution.
Through analysis, it is found that the effective signal of the original data covers 16-bit dynamic range, and the average signal-to-noise ratio is as high as 110, which can be directly superimposed to generate a whole brain projection map.
The high-definition data quality improves the reconstruction speed of neuron morphology by nearly 2 times even when the complexity is increased by 5 times.
The article also gives the results of online quantitative statistics of the whole brain distribution of specific types of neurons, with an average accuracy rate of 95%, indicating that the high-quality raw data obtained by HD-fMOST can support online analysis.
In addition, this technology achieves online lossless compression of 10 TB-level original mouse brain data sets, with a compression rate of 3%, which can be directly written to a U disk or uploaded to the cloud, which is expected to greatly reduce the amount of high-resolution brain three-dimensional data sets.
The burden caused by data storage and transmission.
Figure 2 HD-fMOST results of high-definition three-dimensional imaging of mouse brains with sparsely labeled specific neurons.
In summary, the LiMo microscopy proposed by the team can significantly improve the background suppression ability during fast high-resolution optical imaging.
The HD-fMOST technology developed on this basis not only greatly improves the data quality of whole brain optical imaging, but also opens up a new way to solve the big data problems in the field, in terms of data storage, transmission, processing and analysis.
Efficiency has significantly improved efficiency and is expected to play a huge role in standardized and large-scale brain science research.
Dr.
Zhong Qiuyuan and Professor Li An’an are the co-first authors, Academician Luo Qingming and Professor Yuan Jing are the co-corresponding authors, PhD student Jin Rui, Master Zhang Dejie, Professor Li Xiangning, Master Jia Xueyan, PhD student Zhangheng Ding, Doctor Luo Pan, PhD student Zhou Can, and Chenyu Jiang Master, Dr.
Feng Zhao, Professor Zhang Zhihong, and Professor Gong Hui are co-authors.
Particularly worthy of attention, the journal Nature Methods also reported an interview with Professor Qingming Luo during the same period.
In the interview, Mr.
Luo reviewed the 20-year MOST research process and pointed out that HD-fMOST is not a whim.
Facing the major demand for drawing the whole-brain mesoscopic neural connection map, the whole team has been exploring and researching for a long time.
It is to discover new problems in continuous experiments, and then solve them, promote the results of continuous technological innovation, and there will be more and more achievements in the future.
New methods and new technology output.
Original link: https://doi.
org/10.
1038/s41592-021-01074-x Expert comment Prof.
Xue Tian (Executive Director of the Department of Life Sciences and Medicine, University of Science and Technology of China)'s understanding of life is largely derived from biological samples Microscopic observations, the invention of the microscope and the discovery of cells hundreds of years ago opened up our understanding of life mechanisms.
Many major breakthroughs in the field of life sciences today have come from the development of microscopic observation technology.
The so-called "seeing is believing" is true.
In recent years, the research of brain science has advanced by leaps and bounds.
The brain is the most complex organ, and its sub-micron-level cell structure and connection mode are the material basis for its function.
Therefore, a series of imaging techniques for brain tissue have emerged.
Fluorescence microscopy optical slice tomography (fMOST) technology developed by the team of Academician Luo Qingming of Huazhong University of Science and Technology and continuously updated iteratively is the only one in the world that can be used to achieve whole-brain-scale single-cell connection resolution mesoscopic brain mapping.
Imaging technology, many domestic and foreign brain scientific researches, including the American Brain Project, rely on fMOST technology to achieve whole-brain-scale single-cell brain mapping.
On this basis, the team of Academician Luo Qingming recently continued to innovate from the source imaging technology, and developed a new method of line illumination modulation (LiMo) microscopic imaging, which uses the natural intensity modulation of line illumination to linearly scan the sample, and then through simple One-step linear calculation to reconstruct the optical slice image solves the problem that high resolution, high throughput and high definition cannot be taken into account in the traditional optical microscopy imaging process.
Their team combined LiMo technology with thin-layer tissue sectioning technology to further develop high-definition fluorescence microscopy optical section tomography (HD-fMOST) technology.
This imaging technique has an efficient background effect, allowing the recording and displaying of the rich details of neuronal processes within a wide dynamic range.
HD-fMOST's high signal-to-noise ratio and high-definition imaging data make it easier, more stable, more accurate and more efficient to automatically and manually track neuron morphology, and greatly improve the accuracy and work efficiency of single neuron reconstruction .
This improved imaging quality from the source imaging technology greatly facilitates the analysis and operation of sparse and high-resolution brain imaging data, can achieve more than 30 times lossless data compression, and supports online data storage and analysis.
The results of the article demonstrate the potential of HD-fMOST technology in large-scale acquisition and analysis of high-resolution data sets of the whole brain.
My own research group is very fortunate to use HD-fMOST technology in advance.
It is no exaggeration to say that the quality of whole brain imaging data obtained by HD-fMOST technology is unprecedented.
We have reason to believe that HD-fMOST imaging technology based on LiMo will greatly promote the drawing of brain mesoscopic connection maps.
We also look forward to the team of Academician Qingming Luo to bring newer and stronger imaging technology to brain science research in China and the world.
Plate maker: Eleven
The fine structure of biological tissues is complex and diverse, and how to use optical methods to observe them comprehensively and accurately in three-dimensional space is a recognized problem.
Neurons with complex shapes are the basic functional units of the brain, and how to obtain their complete structure poses a great challenge to the existing technology.
The diameter of the fluorescently labeled neuron cell body is about 10-20 microns, and the diameter of the axons and numerous branch fibers extending from the cell body is only 0.
2-0.
5 microns, and most of them project to different brain regions of the whole brain.
The brightness of the cell body and fiber differs by 2-3 orders of magnitude, and the spatial distribution is often intertwined.
To detect the weak fluorescent signal on axons under the interference of surrounding cells is like observing small stars around the bright sun.
For such situations, traditional optical tomography methods are difficult to achieve.
On March 1, 2021, the team of Academician Qingming Luo of the Biomedical Photonics Function Laboratory of Wuhan Optoelectronics National Research Center, Huazhong University of Science and Technology published an article High-definition imaging using line-illumination modulation microscopy in Nature Methods and developed line illumination Modulation microscopy and high-definition imaging are realized.
Luo Qingming's team proposed a new method of high-definition, high-throughput optical tomography—Line Illumination Modulated Optical Tomography (Line Illumination Microscopy, LiMo).
They cleverly used the Gaussian distribution of the linear illumination light intensity as a natural illumination intensity modulation mode.
With different illumination intensities, only the signal on the focusing surface produces corresponding modulation, while the background signal outside the focusing is not modulated.Therefore, the multi-line detection method can record these signals modulated by different intensities at one time, and only the simplest one-step linear calculation is needed to remove the same out-of-focus background signal and obtain a clear focal plane optical tomographic image.
Compared with classical methods such as laser spot confocal scanning microscopy, two-photon excitation fluorescence microscopy, laser line confocal scanning microscopy and structured light optical tomography, which are widely used in biomedicine, it is proved by theoretical derivation The background signal of LiMo has a faster attenuation coefficient.
This conclusion is also proved by experimental tests.
The signal-to-background ratio of the LiMo method is 1-2 orders of magnitude higher than that of the above-mentioned traditional methods.
This method only needs a simple multi-line detection line illumination light path, overcomes the inherent defects of residual modulation artifacts in traditional structured light illumination imaging, and does not require multiple imaging to obtain the required data, and has line scanning for a large range of samples The advantage of high imaging throughput solves the problem that traditional fluorescence microscopy optical tomography imaging methods cannot take into account high resolution, high throughput and high definition at the same time.
It can be said that the method, whether it is an optical path or an algorithm, fully reflects the beauty of simplicity of technology.
Fig.
1 Schematic diagram of LiMo microscopic imaging principle and performance test results Based on this, Qingming Luo’s team further developed the high-definition fluorescent micro-optical sectioning tomography (HD-fMOST), which transformed the whole brain from optical imaging.
The high resolution is raised to the new standard of high definition.
In recent years, whole-brain optical imaging has brought unprecedented rich details to biomedical research, but it has also produced huge amounts of data difficulties.
In order to solve this problem, researchers mainly focus on the algorithm field to find solutions.
Luo Qingming's team pointed out in a unique way that the fundamental strategy for solving big data problems should be to improve data quality from the source, thereby improving the efficiency of subsequent data processing and analysis.
They used HD-fMOST to perform three-dimensional high-definition two-color imaging of the mouse brain with sparsely labeled neurons, and acquired 12,000 coronal images and cell structure information within 5 days with a resolution of 0.
3 × 0.
3 × 1 micron voxel.
At present, it is the fastest technology for realizing whole-brain optical imaging with similar voxel resolution.
Through analysis, it is found that the effective signal of the original data covers 16-bit dynamic range, and the average signal-to-noise ratio is as high as 110, which can be directly superimposed to generate a whole brain projection map.
The high-definition data quality improves the reconstruction speed of neuron morphology by nearly 2 times even when the complexity is increased by 5 times.
The article also gives the results of online quantitative statistics of the whole brain distribution of specific types of neurons, with an average accuracy rate of 95%, indicating that the high-quality raw data obtained by HD-fMOST can support online analysis.
In addition, this technology achieves online lossless compression of 10 TB-level original mouse brain data sets, with a compression rate of 3%, which can be directly written to a U disk or uploaded to the cloud, which is expected to greatly reduce the amount of high-resolution brain three-dimensional data sets.
The burden caused by data storage and transmission.
Figure 2 HD-fMOST results of high-definition three-dimensional imaging of mouse brains with sparsely labeled specific neurons.
In summary, the LiMo microscopy proposed by the team can significantly improve the background suppression ability during fast high-resolution optical imaging.
The HD-fMOST technology developed on this basis not only greatly improves the data quality of whole brain optical imaging, but also opens up a new way to solve the big data problems in the field, in terms of data storage, transmission, processing and analysis.
Efficiency has significantly improved efficiency and is expected to play a huge role in standardized and large-scale brain science research.
Dr.
Zhong Qiuyuan and Professor Li An’an are the co-first authors, Academician Luo Qingming and Professor Yuan Jing are the co-corresponding authors, PhD student Jin Rui, Master Zhang Dejie, Professor Li Xiangning, Master Jia Xueyan, PhD student Zhangheng Ding, Doctor Luo Pan, PhD student Zhou Can, and Chenyu Jiang Master, Dr.
Feng Zhao, Professor Zhang Zhihong, and Professor Gong Hui are co-authors.
Particularly worthy of attention, the journal Nature Methods also reported an interview with Professor Qingming Luo during the same period.
In the interview, Mr.
Luo reviewed the 20-year MOST research process and pointed out that HD-fMOST is not a whim.
Facing the major demand for drawing the whole-brain mesoscopic neural connection map, the whole team has been exploring and researching for a long time.
It is to discover new problems in continuous experiments, and then solve them, promote the results of continuous technological innovation, and there will be more and more achievements in the future.
New methods and new technology output.
Original link: https://doi.
org/10.
1038/s41592-021-01074-x Expert comment Prof.
Xue Tian (Executive Director of the Department of Life Sciences and Medicine, University of Science and Technology of China)'s understanding of life is largely derived from biological samples Microscopic observations, the invention of the microscope and the discovery of cells hundreds of years ago opened up our understanding of life mechanisms.
Many major breakthroughs in the field of life sciences today have come from the development of microscopic observation technology.
The so-called "seeing is believing" is true.
In recent years, the research of brain science has advanced by leaps and bounds.
The brain is the most complex organ, and its sub-micron-level cell structure and connection mode are the material basis for its function.
Therefore, a series of imaging techniques for brain tissue have emerged.
Fluorescence microscopy optical slice tomography (fMOST) technology developed by the team of Academician Luo Qingming of Huazhong University of Science and Technology and continuously updated iteratively is the only one in the world that can be used to achieve whole-brain-scale single-cell connection resolution mesoscopic brain mapping.
Imaging technology, many domestic and foreign brain scientific researches, including the American Brain Project, rely on fMOST technology to achieve whole-brain-scale single-cell brain mapping.
On this basis, the team of Academician Luo Qingming recently continued to innovate from the source imaging technology, and developed a new method of line illumination modulation (LiMo) microscopic imaging, which uses the natural intensity modulation of line illumination to linearly scan the sample, and then through simple One-step linear calculation to reconstruct the optical slice image solves the problem that high resolution, high throughput and high definition cannot be taken into account in the traditional optical microscopy imaging process.
Their team combined LiMo technology with thin-layer tissue sectioning technology to further develop high-definition fluorescence microscopy optical section tomography (HD-fMOST) technology.
This imaging technique has an efficient background effect, allowing the recording and displaying of the rich details of neuronal processes within a wide dynamic range.
HD-fMOST's high signal-to-noise ratio and high-definition imaging data make it easier, more stable, more accurate and more efficient to automatically and manually track neuron morphology, and greatly improve the accuracy and work efficiency of single neuron reconstruction .
This improved imaging quality from the source imaging technology greatly facilitates the analysis and operation of sparse and high-resolution brain imaging data, can achieve more than 30 times lossless data compression, and supports online data storage and analysis.
The results of the article demonstrate the potential of HD-fMOST technology in large-scale acquisition and analysis of high-resolution data sets of the whole brain.
My own research group is very fortunate to use HD-fMOST technology in advance.
It is no exaggeration to say that the quality of whole brain imaging data obtained by HD-fMOST technology is unprecedented.
We have reason to believe that HD-fMOST imaging technology based on LiMo will greatly promote the drawing of brain mesoscopic connection maps.
We also look forward to the team of Academician Qingming Luo to bring newer and stronger imaging technology to brain science research in China and the world.
Plate maker: Eleven
