FDISCO:an Innovative Tissue Optical Clearing Method For Whole-Organ Imaging

There is a growing trend to visualize the fine tissue structures in three-dimensional(3D)in biological studies,such as neuroscience and systems biology.Optical microscopy can image the tissue structure at subcellular resolution.However,the high scattering characteristic of tissues limits the penetration depth of light and impedes the 3D imaging of tissues by optical microscopy.Recently,tissue optical clearing methods have been emerged to reduce the light scattering and make tissues transparent for 3D optical imaging.Currently,a number of tissue clearing methods in vitro have been developed and widely used for 3D fluorescence imaging of biological tissues,such as the mouse brain.However,the existing tissue clearing methods have some deficiencies on whole-organ clearing and imaging,and there is a lack of clearing method with high level of clearing capability and fluorescence preservation.This study is aimed to develop a new efficient clearing method named FDISCO(DISCO with superior fluorescence-preserving capability),which has the advantage in both clearing effect and fluorescence retention,for whole-organ imaging.(1)The development of new tissue clearing method: Based on the three-dimensional imaging of solvent-cleared organs(3DISCO),the effects of different temperature and pH conditions of clearing agents on the enhanced green fluorescent protein(EGFP)stabilization were investigated to determine the fine conditions for fluorescence retention.These conditions resulted in an advanced tissue-clearing protocol with improved fluorescence preservation,termed FDISCO.And it was proved that FDISCO can not only preserve the fluorescence of various fluorescent proteins well but also have good compatibility with multiple chemical fluorescent tracers.(2)Performance comparison between the FDISCO and other clearing methods: To test the clearing capability,FDISCO was applied for whole-brain clearing and imaging.And the results were compared with those of other existing whole-organ clearing methods.We quantitatively evaluated the performance of clearing and imaging in five aspects: tissue transparency,time to clear,morphological changes,fluorescence level and imaging depth.It was found that FDISCO performed better on tissue clearing and imaging than other methods,and achieved the 3D visualization of whole mouse brain.In addition,FDISCO was proved to be a scalable clearing method achieving effective clearing of various specimens of mouse and rat,including the soft tissue and hard tissue.(3)Neural and vascular imaging of whole organs based on FDISCO: In combination with fluorescence labeling(transgenic mice,virus tracing and fluorescent dyes),FDISCO clearing,and light-sheet fluorescence microscopy(LSFM)imaging,we performed the whole-organ imaging of the nervous and vascular systems in the mouse and rat.The results indicated that FDISCO achieved 3D reconstruction of detailed neuronal and vascular structures throughout the whole organs,such as the brain,spinal cord,muscle and kidney.Moreover,FDISCO achieved a fine repeated imaging of cleared tissues over a year,and had the potential to detect weak fluorescence signals expressed by viruses.In this work,we developed an advanced solvent-based clearing method,named FDISCO,by temperature and pH conditions adjustment based on 3DISCO.FDISCO can achieve a high level of fluorescence preservation of multiple probes while maintaining potent tissue-clearing capability and size reduction.Combined with the FDISCO clearing and LSFM imaging,we obtained the 3D reconstruction of neural and vascular structures in whole organs and confirmed that FDISCO allows the detection of weak fluorescent signals.FDISCO provides an efficient method for 3D visualization and analysis of anatomical structures in large-volume tissue and facilitates the study of the clinical diagnosis and interventions for pathological diseases.