Cofocal Fluorescence Micro-Optical Sectioning Tomography

"As humans we can identify galaxies light years away, we can study particles smaller than an atom, but we still haven’t unlocked the mystery of the three pounds of matter between our ears ". as U.S. President Obama said at a news conference in2013. The structure of brain circuits is very complex and diverse, a simple function often needs many types of neural circuits working together in the brain, so obtaining the structure of neural circuits in the whole brain is the basis of understanding brain function and brain diseases. Fluorescence labeling has become an important tool to label specific brain circuits. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge.Using fluorescentlabel to imageneural connectionsof whole brain at micron resolution is one of the difficulties in three-dimensional fluorescence structureimaging. Although Micro-Optical Sectioning Tomography (MOST) has obtained sub-micron resolution of whole brain mouse atlas,MOST are based on the absorption of wide field reflection imaging; when confronted with large background fluorescence in fluorescence imaging, the spatial resolution and signal-to-noise ratio (SNR) got worse. To solve this problem, this paper proposesto usea confocal way to realize optical tomographyon the knife, to reject background florescent noise of MOST due to high scattering in order toachieve high-resolution of MOST fluorescence imaging.Here, confocal point scanningis used instead of wide field of line scan. As a result, scanning speed decreases significantly.It is bad for large volume imaging. This paperexploredprinciplesof different fast scanused in fluorescent imaging of MOST. Aninertial-free scanner based on acousto-optic principlewaschosen toachieve fast and stable scan.To further improve the scanning speed, this paper put forward expanding scan angleby shrinking beam to increase the field of viewwithout losingof scan speed, which guaranteedfast imaging. Astigmatism appears when acousticoptical scanner worksin fastscan, deteriorating the spatial resolution. This article analyses the cause of astigmatism and itsinfluence on imaging resolution.By using long focal length of cylindrical lens to compensate the astigmatism and reduce the sensitivity and difficulty of adjustment for lens position, submicron optical spatial resolution is ensured.Imaging long projection of single axon with high signal to noise radio (SNR) is antherdifficultyin three-dimensional fluorescence structureimaging. Based on confocal imaging, of fluorescence MOST using uses knife cutting sample and withat the same time simultaneous point scan and detection bythrough slit aperture. This paper analyzes the influence factor of confocal fluorescence MOST, and raisesmethods of increasing signal,and decreasing the excitation and detection of background fluorescence noise. Optimal SNR could be achieved, makeing sure imaging single axon in large volume.Here, signaltonoise ratio reduces because that rapid scanning reduces the signal integration time. To decrease the excitation of background fluorescence noise, three dimensional point spread function (PSF) model was used to describe " imaging over knife edge" imaging structure. It is found out the excitation of background fluorescence can be removed by elevating laser excitation cone to separate the slice from the left sample bulk to some extent, and and find out this structure can remove the excitation of background fluorescence by elevating laser excitation cone and separate the slice from fluorescence bulk.Combined with confocal sectioningdetection, increase SNR and axis resolution can be further improvedbased on confocal imaging. Sectioning the fluorescence sample sometimes induces tears between adjacent tiles and causes difficulties in continuous fiber tracing from fluorescence imaging. Choosing optimized a50-μm-width confocal slit, the signal-to-background ratio is increased16-to49-fold more than that without the slit, which effectively improves the detectability of the signal in the interruptions and enables continuous tracing of the neuronal circuits, to ensure data integrity of large volume.Based on the work discussed above, this paperhas designed and implemented a confocal fluorescence microscopy optical sectioning tomography system (we called Confocal fluorescence Micro-Optical Sectioning Tomography, Confocal-fMOST). This paper designs and implements high stability scan image software based on state machine, accurate synchronization of scan image and cutting movement, does stability design and tests core part of this system.This system could be used to fluorescence imaging the sample volume upto28cm*4.5cm*2cm centimeter cubic with one micron voxel resolution (1μm x1μm×1μm),and continuously work for ten days,24hours per day, implements a complete whole mouse brain circuit within10days.Our System has been successfully used for obtaininga whole brain datasets of Thyl-eYFP-H transgenic mouse. Using the data sets, we have demonstrated the first long-range tracing of individual axons in the mouse brain. The system provides a set of toolsfor exploring the local and long-distance neural circuit. Confocal-fMOST system is shown to be a powerful tool to decipher the whole brain circuits, demonstrating that its potential applications in neuroscience research.