Neuron. Chen Yixuan and others developed ribosome marker neurocytoplasm imaging technology.

The complex structure and signal transmission of neural networks constitute the basis of animal behavior and sense organs.it is necessary to study the interconnected nervous system as a whole to understand its principle.at present, fluorescence imaging has become the mainstream method for observing neural networks: fluorescent proteins can be used to label specific neural tissues and display their structures; fluorescent probes can transform biological signals such as calcium ion concentration into optical signals to show their dynamic state.through fluorescent protein, optical imaging can detect hundreds or even thousands of neurons at the same time. At the same time, it is necessary and difficult to summarize the signal to each neuron.one of the main causes of suffering in this step is the complex structure of nerve cells, each neuron contains two parts: soma and neural processes.this feature leads to the neuronal bodies often buried in the neurites of other nerves, which makes it difficult for individuals to distinguish each other, and the optical signals from different sources interfere with each other.many ways have been proposed and developed to solve this problem.for example, more sophisticated optical instruments such as two-photon imaging can roughly distinguish the boundaries between the cell body and surrounding tissues with a more compact point spread function.but the improvement of accuracy is often accompanied by the sacrifice of speed, and the interference between small nerve tissues can not be completely solved by improving the imaging accuracy.from the computational level, non negative matrix factorization (NMF) is used to divide the neural image into the sum of individuals, which greatly reduces the interference between overlapping signals.however, the assumptions and uncertainties in such calculation functions will vary with experimental conditions and measurement objects, and the results obtained can not be directly verified, resulting in potential over compensation and artificial noise.since the problem of signal decomposition lies in the interference between fluorescent protein in neurite and fluorescent protein in cell body, it should be the most direct method to eliminate the signal from neurite.for example, the use of nuclear localization sequences (NLS) to transfer fluorescent proteins into the nucleus can make neurons clearly defined.however, due to the poor coupling between intracellular calcium signal and cell electrical signal (calcium imaging is usually used to infer electrical signal when measuring neurons), the time accuracy of GCaMP of calcium ion sensor will be significantly reduced.on June 22, 2020, Zachary Knight group of UCSF and Jennifer garrison group of Buck Institute (the first author is Dr. Chen Yiming) published the title "soma targeted imaging of neural circuits by ribosome" in neuron magazine Tethering's paper, using the characteristics of ribosomes, shows a method to localize fluorescent proteins in cells and retain their precision.the signal of a fluorescent protein can be highly aggregated and enhanced by ribose labeling. In the postdoctoral stage, Zack has made a nano-l10 transgenic mouse.the ribosomal subunit L10 in this mouse was labeled by nanobody, a single domain antibody of GFP, through which GFP was accumulated on the ribosome. in the early days of Knight laboratory, the application of this technology mainly focused on extracting ribosomes and mRNA on ribosomes for deep sequencing (TRAP) of specific cells. with the progress of the study, Knight group observed that these GFPs connected to ribosomes were always located in the cell bodies of neurons, and they always made the cells very bright, probably due to aggregation. gradually, we began to use ribosomal linked fluorescent protein to analyze the distribution and quantity of target cells. the beginning of the article shows this process and tests the applicability of the method in a variety of different nerve tissues. it is particularly noteworthy that most of the GFP transgenic mice that produced hundreds of kinds of labeled cells or proteins in the past had weak GFP signals. When these GFPs were aggregated by nano-l10, the signals were significantly enhanced, which made these transgenic plants more widely used and avoided misreading of GFP cells themselves because of the absence of signals Distribution. the ribosomal tagging method can also be applied to GCaMP to improve the quality of calcium imaging. if all GCaMP can be limited to the intracellular, it will be a significant improvement in optical measurement in the field of neurobiology. so the authors directly linked GCaMP with ribosomal subunit L10 and cloned ribo GCaMP. after testing, the authors found that in the cortex, hypothalamus, hippocampus and superior colliculus, ribosomal markers could significantly weaken and almost eliminate GCaMP in neurites. through high-precision two-photon scanning imaging, we compared the efficiency of ribose labeled ribo GCaMP and ordinary GCaMP in reporting action potential in different parts of neurons. in this experiment, it was found that in vivo, the signal intensity of ribo GCaMP and that of ordinary GCaMP were almost the same. However, the signal intensity of ribo GCaMP was almost the same in different parts of neurons. in order to further confirm the accuracy of GCaMP, a high-throughput field current stimulation and single photon recording were used. The sensitivity, time accuracy and SNR of ribo GCaMP and GCaMP are similar under different conditions. triple ribo GCaMP can be used for in vivo calcium imaging in mice and reduce signal noise. Can ribo GCaMP show its advantages under the common optical machines in the field? In mice, the expression of ribo GCaMP was measured by two-photon imaging, micro microscope and GRIN lens. in the visual cortex, the authors were surprised to find that even two-photon imaging suffers from this serious mutual contamination of signal sources. specifically, in the data collected through GCaMP, the closer the neurons are to each other, the higher the correlation coefficient between their signals, which is likely due to the overlap between neurons through neurites. however, when the data were collected with ribo GCaMP under the same experimental conditions, the coupling between the distance and the correlation coefficient almost disappeared (except for the distance within 50 μ m). it can be seen that eliminating GCaMP from neurites can improve the quality of data. however, when the authors observed the medial prefrontal cortex (mPFC) with a single photon micro microscope through a gradient refraction filter, the enhancement became very insignificant. the author thinks that the main signal pollution is the scattering of light and the interference of focusing external light source under the experimental conditions, and limiting the fluorescence in the cell body can not effectively improve these optical deficiencies. finally, in two experiments, the authors found that ribose labeling reduced the brightness of GCaMP. For example, it took about 2.4 times the laser intensity to achieve approximately the same brightness under the two-photon microscope. at present, there is no way to solve this defect, which may be due to the limitation of ribosomal number or the distribution of ribosomes in the sites with low calcium ion concentration. in vivo imaging is very important for the toxicity of the fluorescent protein expressed. the expression of ribo GCaMP and ordinary GCaMP in brain tissue was observed by immunostaining, measurement of cell membrane characteristics and in vivo microscope. the authors did not find any essential difference in cytotoxicity between the two. in conclusion, ribose labeling is an effective method to limit GCaMP in vivo without changing its toxicity and activity. four ribo GCaMP can enhance the whole brain calcium imaging of nematodes. In addition to mice, nematodes are also commonly used model organisms in neurobiology. the authors then tested whether the technique could improve calcium imaging in nematodes by expressing ribo GCaMP. because of the smile of the nematode's head, the activity of all neurons in the whole head can be easily recorded in one microscopic field of vision. in the past, the imaging technology relied on GCaMP (NLS GCaMP) with approved localization sequence to distinguish neurons from complex neural networks. if ribo GCaMP can achieve the same effect without sacrificing time precision, it will be a good technology improvement. after testing, compared with ordinary GCaMP, ribo GCaMP can eliminate signal pollution between neurons and allow whole brain, single neuron resolution calcium imaging. compared with NLS GCaMP, ribo GCaMP can significantly improve the time accuracy of calcium imaging, and can show a lot of neural activities that will be missed by NLS GCaMP. different from the test in mice, ribo GCaMP still retains the brightness of ordinary GCaMP. in this article, the authors demonstrate an easy-to-use and highly efficient technique to eliminate the interference of cell process signals. by connecting soluble fluorescent proteins to ribosomes indirectly or directly, the authors immobilized the signal in the cell body. of course, other sites can also be used for cell targeted immobilization, such as motif of potassium channel 2.1 (Kv2.1), ankyrin, motif that can slow down protein transport, and kainate receptor subunit 2 can more or less fix proteins around the cell body. looking back on the development and testing of this technique, the authors believe that ribosomes have several differences compared with ribosomes, which make some of their properties particularly suitable for the immobilization of soluble proteins. first of all, unlike membrane proteins, ribosomes themselves are soluble, thus ensuring the surrounding water environment and the activity and folding of the trapped proteins. then, ribosomes themselves have not found significant interaction with calcium ions in many studies on ribosomes, which may explain why there is no cytotoxicity in connecting GCaMP to ribosomes. at the same time, as the largest number of organelles, ribosomes can provide a large number of fixed sites to ensure that a certain number of trapped proteins can produce effects. finally, ribosomal protein, as a very long-standing and important cell component, has a set of tight regulatory mechanisms. for example,