MACS: Rapid Aqueous Tissue Clearing Method For 3D Imaging Of Intact Organs

In recent years,more and more studies have been conducted on whole organs and organisms.Therefore,three-dimensional(3D)imaging of tissue structures with high resolution becomes rather important for biomedical research.The combination of advanced optical imaging techniques and a variety of fluorescent labeling techniques provides an essential tool for acquiring 3D information of tissues.However,the high scattering within tissues limit the imaging depth of optical temography.Tissue optical clearing technology can reduce the light scattering and makes the tissue become transparent,thus it can deepen the optical imaging depth and improve the imaging quality and provide new insight for 3D imaging of large-volume tissues.At present,there are two kinds of tissue clearing methods,namely solvent-based methods and aqueous-based methods.The former can provide high transparency but suffer from fluorescence quenching.The latter can preserve fluorescent signals and tissue morphology well,but they are faced with long transparency time,insufficient transparency and complex handling.In addition,in order to obtain high transparency,many of the clearing methods use high concentration organic solvents or detergents,which makes them incompatible with commonly-used lipophophilic dyes.These drawbacks limit the application of tissue clearing methods.This study is focused on solving the above problems,aiming to develop a new aqueousbased method which simultaneously possesses fast clearing speed,high transparency,well fluorescence compatibility and easy operation.The main contents are as follows:(1)Establishment of the new aqueous-based method: Based on the basic chemical principle of tissue clearing,new clearing reagents were widely screened.The chemical properties of reagents including structure,water-solubility,refractive index and viscosity were considered in preliminary screening.M-xylylenediamine(MXDA)was found to have great potential and was first introduced in tissue clearing.The optimal concentration of MXDA was determined for both transparency and fluorescence preservation.Sorbitol was added to further enhance the clearing performance.In addition,the tri-step protocol was designed to enhance the clearing of whole adult mouse organs.Therefore,a new aqueousbased method with hight clearing performance,termed MACS(M-xylylenediamine based Aqueous Clearing System),was established.(2)Comparion of clearing performance between MACS and other methods: MACS was applied to clear various tissues and was compared with other clearing methods in terms of clearing time,transparency,tissue size change and fluorescence preservation.MACS achieves high transparency on whole adult mouse brain in 2.5 days,which is faster than the current solvent-based methods(at least 3 days)and near four times faster than the commonly used aqueous-based methods(e.g.CUBIC).More importantly,MACS effectively maintains the membrane structure of tissues,thus perform perfect compatibility with lipophilic dye DiI.In addition,MACS can also be applied to clear a variety of rodent organs and even the whole mouse body.During clearing,MACS can effectively remove the residual blood and reduce the absorption within tissues(3)MACS is capable for 3D imaging of neural structures in intact organs: By combining with a variety of fluorescence labeling techniques(transgenic labeling,immunostaining labeling,and virus labeling),3D reconstruction of neural networks of whole mouse organs and embryos were performed via MACS and light sheet imaging.MACS can not only clearing and imaging mouse brains which are transgenic-labelled and virus-labelled virus,more importantly,by combining with MXDA pretreatment to enhance the permeability of antibodies,MACS can also perform whole-mount immunostaining of mouse embryos and imaging the neural structures of embryos at different ages.(4)MACS is capable for 3D imaging of vasculatures in intact organs with DiI labeling:DiI was used to label the vasculature in whole mouse organs and was proved to have better labeling efficiency and integrity in specific organs over several commonly used labeling methods.Furthermore,3D imaging and reconstruction of the vascular network in multiple mouse organs was performed,including the brain,spinal cord,heart,kidney,spleen and small intestine.In addition,in combination with the type I diabetes model,3D visualization of vasculatures in kidney from normal and diabetic mice was performed,and 3D segmentation and quantitative analysis for glomeruli were conducted.The changes in the number,volume and volume distribution of glomeruli were also analyzed.In summary,MACS not only overcomes the drawbacks of current aquous-based methods including slow clearing speed,poor transparency and complex handling,more importantly,MACS successfully achieved both high transparency and excellent compatibility of lipophilic dyes DiI.MACS could provide a valuable alternative for the clearing,labeling,and imaging of large volume tissues and facilitate diagnostic studies for pathological diseases.