Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.
Three-dimensional mapping of skeletal muscle is particularly valuable for systematical identification and analysis of various biological components, such as nerve fibers, muscle fibers and vessels during disease and regeneration. iDISCO, as a whole-mount immunolabeling technique, provides an important tool for volume imaging of muscles, but its application has been limited to thin and small muscles of young mice owing to poor antibody penetration. In this work, we developed a modified iDISCO method and applied it to label the nerve fibers in various skeletal muscles of adult mice, including diaphragm, biceps brachii and tibialis anterior. Light-sheet microscopy was used to image biceps brachii and tibialis anterior. The results showed that the modified method could achieve uniform and complete labeling of nerve fibers within muscles, whereas original iDISCO method caused strong nonspecific signal in the surface of the muscles, and intramuscular nerve fibers were almost invisible. The modified method permitted us to trace three-dimensional nerve fibers in the muscles. This method shows potential for three-dimensional histological analysis in mouse skeletal muscle, facilitating the understanding of structural-functional relationship of skeletal muscle in physiological and pathological condition.
Recently, a variety of tissue optical clearing techniques have been developed to reduce light scattering for imaging deeper and three-dimensional reconstruction of tissue structures. Combined with optical imaging techniques and diverse labeling methods, these clearing methods have significantly promoted the development of neuroscience. Each of them has its own characteristics with certain advantages and disadvantages. Though there are some comparison results, the clearing methods covered are limited and the evaluation indices lack uniformity, which made it difficult to select a best-fit protocol from numerous methods for clearing in practical applications. Hence, it is necessary to systematically assess and compare these clearing methods. We evaluated the performance of seven typical clearing methods, including 3-D imaging of solvent-cleared organs (3DISCO), ultimate DISCO (uDISCO), see deep brain (SeeDB), ScaleS, ClearT2, clear, unobstructed brain imaging cocktails and computational analysis, and passive CLARITY technique (PACT), on mouse brain samples. First, we compared the clearing effect and clearing time as well as size deformation on brain tissues. Further, we evaluated the fluorescence preservation and the increase of imaging depth induced by different methods. The results showed that 3DISCO, uDISCO, and PACT possessed excellent clearing capability on mouse brains, ScaleS and SeeDB rendered moderate transparency, whereas ClearT2 performed the worst. uDISCO and 3DISCO induced substantial size reduction on brain sections, and PACT expanded the mouse brain most seriously. Among those methods, ScaleS performed best on fluorescence retention, 3DISCO induced the biggest decline of the fluorescence. PACT achieved the highest increase of imaging depth, and SeeDB and ClearT2 possessed the shallowest imaging depth. This study is expected to provide important reference for users in choosing the most suitable brain optical clearing method.
Light-sheet fluorescence microscopy (LSFM) uses an additional laser-sheet to illuminate selective planes of the sample, thereby enabling three-dimensional imaging at high spatial-temporal resolution. These advantages make LSFM a promising tool for high-quality brain visualization. However, even by the use of LSFM, the spatial resolution remains insufficient to resolve the neural structures across a mesoscale whole mouse brain in three dimensions. At the same time, the thick-tissue scattering prevents a clear observation from the deep of brain. Here we use multi-view LSFM strategy to solve this challenge, surpassing the resolution limit of standard light-sheet microscope under a large field-of-view (FOV). As demonstrated by the imaging of optically-cleared mouse brain labelled with thy1-GFP, we achieve a brain-wide, isotropic cellular resolution of ~3μm. Besides the resolution enhancement, multi-view braining imaging can also recover complete signals from deep tissue scattering and attenuation. The identification of long distance neural projections across encephalic regions can be identified and annotated as a result.
The emergence of various optical clearing methods provides a great potential for imaging deep inside tissues by combining with multiple-labelling and microscopic imaging techniques. They were generally developed for specific imaging demand thus presented some non-negligible limitations such as long incubation time, tissue deformation, fluorescence quenching, incompatibility with immunostaining or lipophilic tracers. In this study, we developed a rapid and versatile clearing method, termed ReagentTF, for deep imaging of various fluorescent samples. This method can not only efficiently clear embryos, neonatal whole-brains and adult thick brain sections by simple immersion in aqueous mixtures with minimal volume change, but also can preserve fluorescence of various fluorescent proteins and simultaneously be compatible with immunostaining and lipophilic neuronal dyes. We demonstrate the effectiveness of this method in reconstructing the cell distributions of mouse hippocampus, visualizing the neural projection from CA1 (Cornu Ammonis 1) to HDB (nucleus of the horizontal limb of the diagonal band), and observing the growth of forelimb plexus in whole-mount embryos. These results suggest that ReagentTF is useful for large-volume imaging and will be an option for the deep imaging of biological tissues.