Novel fluorescent tools and techniques for 3D imaging of the cleared brain

Abstract

Background: To better understand the complexity of the brain and how it becomes impaired under different pathological states, a considerably large number of brains would be needed for imaging to generate highly detailed maps in 3D. Chemical probes can offer a readily scalable labelling method that is robust, easy to use with the quick operation, and feasible for human tissue where genetic viral and toxin tracers are inappropriate. The drawbacks of immunostaining methods have spurred interest in developing alternative strategies to visualize the optically transparent brain, especially from fixed archived samples or human autopsies that are not optimally fixed.

Purpose: We envision AIE-based probes and techniques as robust tools when paired with clearing methods for visualizing the human brain. This thesis aims to develop alternative strategies to tissue labelling using novel AIE-based fluorescent chemical probes and methods that offer easy operation, high brightness, photostability and contrast suitable for 3D visualization of neurons and nerve fibers in mouse brains.

Paper I: The novel water-soluble silver-ion sensitive AIE probe TPE-4TA achieved by tetrazole-Ag+ coordination, allowed for the development of a new fluorescent silver (silver-AIE) method to visualize separated proteins following sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Compared with conventional silver nitrate stains, silver-AIE not only offers sensitive fluorogenic detection of proteins, but it is quantifiable, easy to use, has a broad linear dynamic range and a great contrast which rivals the popular commercial stain, SYPRO Ruby. Study II describes how to troubleshoot the fluorescent silver gel stain, alternative steps for rapid staining and techniques to carry out the procedure correctly to avoid suboptimal results.

Paper II: We report a novel fluorescent silver stain for fixed mouse brain tissue compatible with multiplexed immunofluorescence imaging in paraffin sections. The Ag+-specific aggregation-induced emission (AIE) strategy outperforms the chromogenic detection employed by many conventional silver staining protocols to visualize neurites and fiber tracts in paraffin sections or passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / in situ-hybridization-compatible Tissue hYdrogel (CLARITY) -cleared tissue. This enables imaging using standard fluorescent widefield or optical sectioning microscopies. Not only does our method uses less hazardous reagents, but the highly sensitive TPE-4TA also uses silver nitrate concentrations up to two million-fold lower than the standard Yamamoto-Hirano’s modification of the Bielschowsky stain.

Paper III: Development of the novel near-infrared AIE fluorescent probe PM-ML with D-π-A (donor-pi-acceptor) structure for the selective staining of myelinated fibers in the teased sciatic nerves, mouse brain cryosections and ClearT-cleared mouse brain tissue for 3D fluorescent imaging. We envision PM-ML as a potential tool for studying demyelination and evaluated its selectivity, photostability and signal-to-background (SBR) ratio which outperformed common commercial fluorescent myelin staining dyes.