Abstract
Extensive mapping of neuronal connections in the central nervous system requires high-throughput µm-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multi-modal optical techniques. Here, we introduce a versatile brain clearing agent (2,2′-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.
Highlights
Extensive mapping of neuronal connections in the central nervous system requires high-throughput mm-scale imaging of large volumes
To perform two-photon fluorescence microscopy (TPFM) imaging we selected 47% TDE/phosphate-buffered saline (PBS) solution which corresponded to the refractive index (RI) matched by our microscope objective (Zeiss 20X Scale objective, with n 5 1.42)
In this work we presented a simple, quick and inexpensive clearing method based on TDE as refractive index matching agent
Summary
Extensive mapping of neuronal connections in the central nervous system requires high-throughput mm-scale imaging of large volumes. In LSM the sample is illuminated with a thin sheet of light confined into the focal plane of the detection objective, which collects the fluorescence emission along an axis perpendicular to the illumination plane[6] This technique drastically reduces the imaging acquisition time and achieves excellent resolution at high penetration depths; it requires the sample to be transparent. To reduce scattering and to make the tissue transparent the refractive index has to be homogenized inside the sample To this end different approaches have been developed. High refractive index organic solvents have been used for optical clearing of entire mouse brains[7,8,9] These methods guarantee high transparency, protein fluorescence quenching and tissue shrinkage limit their applicability. Presents other nonnegligible limitations such as long incubation times, structural alteration and incompatibility with immunostaining
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