A versatile clearing agent for multi-modal brain imaging

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.

Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging

“…NIR reflectance could be used in combination with serial two-photon microscopy [19,20] for mapping axonal projections over large regions, like the entire hippocampus. The BS signal also helped in recognizing characteristic anatomical features of the tissue, thus making it a useful tool for brain atlassing.…”

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains

“…Crucially for the high-throughput imaging of large samples that we aim for, the protocol, however, is also fast, easy, and cheap. We therefore, developed a versatile, simple, rapid, and inexpensive clearing method described elsewhere, 13 which in combination with CLARITY allows for the optical clearing of entire mouse brains or portions of human tissue.…”

“…The clarified brains are imaged and immersed in clearing solution composed of 63% 2,2′-thiodiethanol (TDE) in phosphate-buffered saline (PBS) and a refractive index of n ≈ 1.45. 13 To fill the entire volume of the sample chamber, ∼150 ml clearing solution is needed. A motorized x-, y-, z-, θ-stage (see Table 1) allows free 3-D motion and rotation of a Teflon cylinder, which reaches into the centre of the chamber [ Fig.…”

“…13 Adult mice (postnatal day 56) were anaesthetized with isoflurane and transcardially perfused with 20 ml icecold PBS solution (pH 7.6) followed by 20 ml of a mixture of 4% (wt/vol) paraformaldehyde (PFA), 4% (wt/vol) acrylamide, 0.05% (wt/vol) bis-acrylamide, 0.25% (wt/vol) VA044 in PBS. We then extracted the brains from the skull and put them in the same PFA/acrylamide/bis-acrylamide/VA044/PBS solution at 4°C for 3 days.…”

Clearing of fixed tissue: a review from a microscopist’s perspective

“…5,6,[8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Here, we try to classify clearing approaches based on the microscopy technique where they can be applied, rather than on the clearing mechanism. 7 We believe that this new taxonomy can be a helpful starting point for microscopists willing to work with transparent tissue.…”