Advancing Optical Tissue Clearing Techniques For Whole-Body High-Resolution Light Sheet Imaging

Sammanfattning

Light sheet microscopy allows for whole-body three dimensional imagingof biological samples. However, penetration depth of the light as well asresolution and quality is limited due to light scattering and absorption inthe sample. Optical tissue clearing methods have been developed for manyyears to reduce the amount of scattering and absorption by creating a uniformrefractive index throughout the sample and reducing the amount of pigmentsin the sample.This thesis evaluated established protocols for tissue clearing (2ECi, iDISCO+and PEGASOS) and based on these results developed new protocols byintroducing different treatment steps to target the specific need of differentbiological samples (rat embryos and zebrafish). Due to the toxic nature ofmany clearing agents and their incompatibility with microscope components,all new protocols used ethyl cinnamate as the refractive index matchingreagent.Protocols were evaluated through visual inspection, imaging with both lightsheet microscopy and confocal microscopy, and image quality was quantifiedusing FRC-QE and signal intensity. All established protocols were not fullyeffective in clearing the whole body due to the highly pigmented and lipid-richnature of the samples. Delipidation and depigmentation were the main focus ofthe new protocols executed on the rats. Methanol-based series for delipidation,as well as dichloromethane (DCM) showed a lot of promise in removing thelipids. Depigmentation with hydrogen peroxide was as well effective butresulted in many air bubbles, reducing image quality. Depigmentation alsobecame a crucial step in clearing the zebrafish due to the highly pigmentedskin. Here as well, air bubbles became the biggest obstacle. Differentmethods of bleaching were developed, by experimenting with different ratios,temperatures and times.All new protocols showed a lot of promise with some protocols having betterimage quality than others. However, further research is needed to fullyoptimize the methods. Finding a bleaching protocol which reduces air bubblesand keeping structure integrity intact while bleaching remains essential inorder to reach full transparency. Additionally, new ways to quantify the resultsare needed.