Light illuminates the "source of imagination": the secret of neuron visualization

From baby babbling, toddler to laughing and scolding, experiencing the ups and downs of life beating, all of them do not require the participation of the bra.
As a vehicle for emotions, thoughts, actions, e.
, the brain is the most fascinating and complex object of study in the univer.
If there is a place for "imagination" to stop, the brain is undoubtedly the orig.
However, how memories are formed and stored, what consciousness is, and how emotions are generated—the occurrences of these everyday events that we take for granted all hold important scientific questions in the field of neuroscien.
From the 17th century when Hooke first observed plant cells through a microscope to the 19th century when Schleiden and Schwan established the cell theory, people gradually realized that the cell is the basic unit of the structure and function of an organi.
However, little is known about the basic components of the nervous system due to the lack of efficient staining methods to "reveal" the nervous syst.
At that time, the scientific community generally accepted the reticular theory proposed by Joseph von Gerlach based on his gold chloride staining method, that is, like the vascular network of the human body, neural tissue is a continuous network formed by the fusion of nerve cel.
In 1873, Camillo Golgi invented the silver dichromate staining method (later called Golgi staining), which can stain a small number of neurons (1-5%), thereby exposing the complete neuronal morphology to the wor.
In the eyes - yet Gorky did not doubt the validity of the reticular theo.
In 1887, Santiago Ramón y Cajal first came into contact with Golgi-stained neural tissue samples and was stunned - he has since refined a large number of brain tissue sections based on a modified Golgi-staining meth.
's observations challenge the reticular theory, pointing out that nerve cells are independent uni.
On the basis of a series of discoveries by Cajal, in 1891, Wilhelm von Waldyer formally proposed the concept of "neuron" and established the neuron theo.
In 1906, Gorky and Cajal were jointly awarded the Nobel Prize in Physiology or Medicine for their outstanding contributions to the analysis of the structure of the nervous syst.
Even at the awards ceremony, the two went head-to-head, debating their own doctrin.
It was not until the invention of the electron microscope that this academic debate was truly finalized - the neuron theory became the basic theory of the development of modern neuroscien.
Cajal is thus considered the father of modern neuroscien.
 Camilo Gorky and Santiago Ramon Cajal (left) and Cajal's drawing of neurons (right) We now know that it is the tiny neurons in the brain that connect with each other through their processes to make up complex nerves The network processes the complex information from inside and outside the body, closely monitors and guides the normal operation of the bo.
It is estimated that the mammalian brain has millions of cells, and the human brain has 86 billion neuro.
Crick and Jones, co-discoverers of the DNA double helix and Nobel Prize winners in physiology or medicine, wrote in a comment to Nature: "I cannot bear that we have not mapped the connections of the human bra.
Without it, there is little hop.
To understand how our brains wo.
" - To understand how the brain works, we cannot escape from the detailed analysis of the structure of the bra.
As the basic unit for studying brain function, the understanding of the structure of neurons will undoubtedly help to understand the information organization structure of the brain - just as we can only grasp the TV set by understanding how each component inside the TV is connected and organiz.
How to play images and sounds, and how to accept commands from the outside wor.
As a star model animal in the field of life science, the analysis of its whole-brain neuron structure has naturally become the first scientific issue that scientists pay attention .
So, as with Cajal, the problem we face is choosing an appropriate technique for visualizing neuro.
A typical neuron consists of a cell body, dendrites, and axo.
Among them, the dendrites are thick and dense, located near the cell body, and receive information input from other neuro.
The axon is long and thin, and can extend very f.
The end of the axon can form a presynaptic terminal, form a synaptic connection with the dendrite or cell body of another neuron, and participate in the interaction between neurons by releasing synaptic transmitte.
information transf.
The neuron cell body usually ranges from tens to tens of microns, but the length of the axon can reach the centimeter level, while its diameter is only 2-5 micro.

That is to say, in order to see the axonal direction of neurons, we should at least choose imaging methods that reach the micron lev.

At present, electron microscopy, as the technical means with the highest imaging resolution, can achieve nano-scale imaging, and has also been applied to the study of neuronal connection maps of small animals such as nematodes and fruit flies - but the brain volume of mice alone is equivalent to 10 million times the size of a worm's brain, let alone a primate with a larger bra.

Considering the cost, time, and volume of data generated for whole-brain imaging in mice - electron microscopy imaging is clearly not applicab.
To resolve neuronal connectivity maps at the whole-brain scale, scientists turned their attention to optical imagi.

 Comparison of brain volume between nematodes, fruit flies and mice dr.

They stained mouse brains using a modified Golgi staining method and coated the mouse brains with Spurr res.

In order to bypass the imaging depth limitation of optical imaging, they creatively proposed a strategy of "imaging while slicin.

During imaging, the fixed mouse brain was sliced ​​into strips of tissue 1 micron thick and 450 microns wide with a diamond blade, and the sliced ​​slices were then imaged by an optical microsco.

After 242 hours, the imaging of the whole mouse brain is complet.

Through the later data processing, the sliced ​​images can be spliced ​​and finally reconstructed to form a three-dimensional mouse whole brain atl.

 Schematic diagram of the imaging principle of MOST technology (left) and mouse brain imaging results (right) Although MOST technology has achieved neuronal imaging of the whole mouse brain, in actual research, scientists prefer to use fluorescent proteins to label certain neuro.

rather than using staining methods to stain whole-brain neuro.

Therefore, the development of MOST technology that can realize fluorescence imaging, namely fMOST (fluorescence micro-optical sectioning tomography), has become their next go.

Different from the brightfield imaging required by dyes, fluorescent proteins are more sensitive to temperature and .

A little carelessness may lead to signal quenching, and the requirements for imaging are even high.
To this end, they improved the processing and coating method of the mouse brain before imaging to minimize the loss of fluorescence signal, while ensuring that the tissue slices have the appropriate hardness to avoid the deformation of the tissue too thin and affect the imaging effe.

In order to accurately obtain the positioning information of neuronal cell bodies and processes in the brain, they also used the landmark positions in the brain combined with MRI and mouse standard brain atlas to perform corresponding calculations and transformations to complete the annotation of neuron morpholo.

——Just like we are in an unfamiliar city, we can easily locate the current position according to the mobile phone map——The two-dimensional standard brain map is equivalent to the mobile phone m.

We combine the landmark buildings we see to realize the mobile phone map and Correspondence to the actual situati.

In 2015, scientists applied stage-scanning line confocal microscopy to further improve imaging spe.

In 2016, based on a fully automated microscopic imaging method called the Brain-wide Positioning System (BPS), they achieved single-cell resolution by simultaneously staining cellular structural markers during imagi.

Precise positioning of cell structur.

In 2021, Luo Qingming's team proposed a new high-definition, high-throughput optical tomography microscopy imaging method-Line illumination modulation optical tomography (LiMo), which can be used in fast high-resolution optical imagi.

Significantly improved background suppression capability greatly improves imaging quality and improves the efficiency of data storage and analys.

 fMOST imaging process demonstration According to reports, it took Pr.

Luo Qingming's team three years to overcome the problem of brain sample preparati.

The research and development of technology involves the cooperation and intersection of multiple disciplines such as biology, optics, machinery, and computer data processi.
After more than ten years of research and development and optimization, fMOST technology has the advantages of high resolution, high throughput, and high definition, and automatic imaging can quickly, efficiently and accurately obtain neuronal morphological information, which can be used for subsequent reconstruction and analysis of neurons in the whole bra.

Metamorphism has laid a solid foundation and has become a powerful tool for the study and analysis of the whole-brain mesoscopic neuron connection map, which is widely used all over the wor.

Similar to human society, each person is an independent individual, but is divided into various groups due to some similar characteristi.

Neurons also possess a range of signatures, gene expression, neuronal morphology, projection information, and electrophysiological properti.

In order to gain a clearer understanding of how the neuronal world works, higher-resolution information on cell populations has become particularly important within neurobiolo.

The clustering information obtained by means of higher resolution helps us to capture those clusters with known projections and functions, and more importantly, to reveal potential new cell populations, and to reveal more systematically how the brain wor.

The development of fMOST and its supporting analytical tools provides us with the possibility to map the brain connection network, and combine with other technical means to build a standard framework of neuron types to help reveal brain functi.

At present, the establishment of a whole-brain neuron connection map library is an important foundation for countries around the world to build global brain science cente.

Now I will share with you in detail how fMOST technology contributes to brain resear.

fMOST collaborates with multi-technology to build a single neuron-level whole-brain connection map One of the main uses of fMOST is to build a whole-brain connection map, thereby collaborating with multi-technology to reveal high-resolution neuronal groupings and help us better explain how the brain functions un.
The research group of Yan Jun and Xu Ninglong of the Center for Excellence in Brain Science and Intelligent Technology Innovation of the Chinese Academy of Sciences cooperated with the Suzhou Brain Spatial Information Research Institute of Huazhong University of Science and Technology and the Gong Hui team of the Wuhan National Research Center of Optoelectronics to publish an online cover article in the journal Nature Neuroscien.

A research paper titled Single-neuron projectome of mouse prefrontal cortex, which nicely demonstrates this application of fMOST technolo.

The prefrontal cortex plays an important role in high-level cognitive functions such as decision-making, working memory, and attenti.

Abnormalities in its structure and function can lead to a variety of brain diseas.

It has a wide range of projections, covering almost most brain regions, including cortex, striatum, thalamus, midbrain, and hindbra.

This article uses the self-developed Fast Neurite Tracer (FNT) optical imaging big data neuron tracking and analysis software to systematically reconstruct the whole-brain projection map of 6357 single neurons in the mouse prefrontal cortex, establishing the world's largest single-neuron projection m.

The mouse whole-brain mesoscopic neural connection atlas database (Figure A); revealed the spatial distribution of 64 neuronal projection subtypes in the mouse prefrontal cortex, and found that there are different projection characteristics between different groups (Figure B) ; elucidated the modular connectivity network and hierarchical structure within the prefrontal lobe (Panel .

While constructing the whole-brain connection network, the researchers are also working with multi-technical collaboration to establish a standardized neuronal typing framework that can integrate multiple molecular-spatial genetic information and multi-faceted phenotypic properti.

The article integrates neural circuit connections with transcriptome cell typ.

The correspondence between neuronal transcriptome subtypes and projection subtypes was obtained by reverse tracing and fluorescence in situ hybridization (Panel .

Previous studies have used population tracer technology to study the whole-brain projection pattern of neurons in the prefrontal cortex, but have not been able to analyze the working pattern and hierarchical structure of the prefrontal brain connection network we.
Single-cell-level imaging enabled by fMOST technology has helped to reveal the regularities of internal connections and external projections in the prefrontal cortex, and suggested a possible working model of the prefrontal cort.

(A) Spatial distribution of reconstructed mPFC neurons in the bra.

(B) Whole-brain axonal projection distribution of mPFC (right), several neuronal subtypes with different projection selectivity (lef.

(C) The results of clustering analysis of single neuron projection maps using the FNT algorithm (left), and the 3D reconstruction results of two differentially high subtypes (SCm-projecting / PCG-projecting) (righ.

(D Modular and hierarchical structure of the intraprefrontal connectivity netwo.

(E) Relationship between prefrontal projection subtypes and genetic subtypes (CT cortical thalamic neurons in PL-ORB brain region.

 fMOST delineates fine neuronal morphology in single cel.

Horizontal neuron fine structure analysis can intuitively reveal the characteristics of neuro.

fMOST can reconstruct neuron morphology at extremely high resolution and observe the fine structure of neuro.

The MOST team led by Professor Luo Qingming of Wuhan National Research Center for Optoelectronics and the United States The research group of Pr.

.

Josh Huang of Cold Spring Harbor Laboratory published in Cell Reports titled Genetic Single Neuron Anatomy Reveals Fine Granularity of Cortical Axo-Axonic Cel.

The fine structure of axonal cells), developed a specific application of fMOST in this rega.
In this article, the researchers designed a fMOST imaging-based, genetically-specific labeling single-cell anatomy platform (gSN.

By solving the four difficulties of single-cell genetic labeling, whole-brain precise imaging, neuronal remodeling and quantitative analysis, the single-cell-scale analysis of AAC (Axo-Axonic Cells) cells has been achiev.

A systematic single-neuron reconstruction of AAC cells in multiple brain regions of the whole brain revealed that the location of AAC cells and the spatial distribution pattern of dendrites and axon fibers were not random, but correlated with cerebral cortical layeri.

By synthesizing a large number of reconstructed cell data, using reasonable quantitative methods and cluster analysis to classify cells, it was found that there are many subtypes of AAC cel.

This study comprehensively analyzed the morphological typing of AAC cells using quantitative and qualitative methods, updated people's cognition of AAC cells, and contributed to further understanding and functional disclosure of AAC cel.

 Technical route and classification results of AAC subgroups in thecortexEvery development of life sciences is premised on the progress of major technologi.

fMOST technology has made significant contributions to the development of brain science, greatly advancing the understanding of brain structure and function, from "seeing" to seeing, to "seeing" and "seein.

Compared with the currently used fMOST imaging technology, electron microscopy can well reconstruct the microscopic structure of cells at nanoscale resolution, providing a state close to the real intercellular connection, and the complete connection map at nanoscale/synaptic resolution provides All information that contributes to the lo.
This knowledge can be used to characterize and model circuits in normal and diseased states, identify functionally relevant key neurons, and discover new cell types based on long-range connectivity and ultrastructural morpholo.

Studies in model organisms such as.

elegans and Drosophila have demonstrated the potential of whole-brain connectivity profiles to support in-depth studies of neural circui.

However, the current electron microscope technology can only reconstruct the structure of ~1 mm³, while the overall level of the mouse brain is about 500 mm³, and the human brain is 1000 times larger than the mou.

At the same time, the technical level requirements for subsequent data segmentation, processing, storage, and computing have also been greatly improv.

But there is no doubt that obtaining nanoscale neuronal connection profile data will cause subversive changes in the neurological field, and is also one of the development goals of subsequent brain mappi.

The refinement of the Atlas program will lay the foundation for the field of neuroscien.

Brain-wide connection profiling and precise mapping of intercellular connections will revolutionize the way brain function is studied, from hypothesis-driven studies to large-scale collaborative projects aimed at elucidating the fundamental rules of functional circui.

These advances will lay the foundation for the discovery of new circuit mechanisms associated with brain diseases, helping to solve the puzzle of brain diseas.
 References:Abbott LF, Bock DD, Callaway EM, et .

The mind of a mouse[.

Cell, 2020, 182(6): 1372-137 Li A, Gong H, Zhang B, et a.

Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain[.

Science, 2010, 330(6009): 1404-140 Gong H, Zeng S, Yan C, et .

Continuously tracing brain- wide long-distance axonal projections in mice at a one-micron voxel resolution[.

Neuroimage, 2013, 74: 87-9 Yang T, Zheng T, Shang Z, et .

Rapid imaging of large tissues using high-resolution stage-scanning microscopy[.

Biomedical optics express, 2015, 6(5): 1867-187 Gong H, Xu D, Yuan J, et .

High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic Landmarks at the cellular level[.

Nature communications, 2016, 7(1): 1-1 Zhong Q, Li A, Jin R, et .

High-definition imaging using line-illumination modulation microscopy[.

Nature Methods, 2021, 18(3): 309-31 Gao L, Liu S, Gou L, et .

Single-neuron projectome of mouse prefrontal cortex[J.

Nature Neuroscience, 2022: 1-1 Wang X, Tucciarone J, Jiang S, et .

Genetic single neuron anatomy reveals fine granularity of cortical axo-axonic cells[.

Cell reports, 2019, 26(11): 3145-315 Source: Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences