Evaluation and Development of Tissue Clearing Protocols For Imaging Large Biological Samples with Light Sheet Microscopy

Sammanfattning

Optical imaging of biological samples is limited by light scattering andabsorption, which reduce penetration depth and image quality. Tissueclearing protocols have been developed to address these issues by removingpigments and reducing refractive index mismatches. This makes it possibleto image larger samples in three dimensions without the need for physicalsectioning.This project evaluated how well existing clearing protocols (2ECi, PEGASOS,iDISCO+) perform on strongly pigmented rat embryos and adult zebrafish,and tested custom protocol variants combining bleaching, pretreatments, andstaining. Performance was evaluated by visual inspection, light sheet andconfocal microscopy, and quantitative analysis (FRC-QE and z-intensityprofiles).The results showed that existing protocols were not fully effective in ratembryos, with strong pigmentation remaining in blood-rich tissues such asliver. Fast Green provided clear structural contrast in light sheet images.Bleaching was the most effective step for improving transparency, especiallyin liver, and also enhanced staining. However, it introduced air bubblesthat strongly reduced image quality. Shorter methanol-based bleaching atlow temperature reduced bubble formation compared to longer PBS-basedbleaching. Vacuum treatment partly restored image quality by reducingair bubbles. These observations were supported by quantitative analysis,although differences in imaging conditions made the results difficult tocompare directly. In zebrafish, bleaching improved transparency, whereasEDTA decalcification had little effect in the tested regions and CUBIC-Lalone was not sufficient to remove pigments.Overall, bleaching emerged as the most critical factor for pigment removal inboth rat embryos and zebrafish, but the conditions still require optimizationto balance transparency with tissue preservation.