Title: Confocal Raman Analysis of Dimethyl Sulfoxide Diffusion in Zebrafish Embryos

Program: Chemistry MS

Committee Chair: Konrad Meister

Committee: Konrad Meister, Oliviero Andreussi, Sophia Theodossiou

Abstract: Dimethyl sulfoxide (DMSO) is widely used as a cryoprotective agent, yet its spatial penetration through protective biological barriers and into embryonic tissue remains difficult to evaluate directly. In this work, confocal Raman microscopy was used to investigate the distribution of deuterated dimethyl sulfoxide, DMSO-d6, in zebrafish embryos embedded in a gel matrix containing 20% v/v DMSO-d6. Zebrafish embryos provide a useful model system because the chorion represents an external diffusion barrier, while the embryo itself contains chemically heterogeneous biological tissue with depth-dependent optical and spectroscopic complexity.
Three-dimensional Raman datasets were collected as z-stacks and processed using a custom Python analysis pipeline developed to quantify and visualize chemical signal distributions throughout the embryo, chorion, and surrounding matrix. DMSO-d6 signal was evaluated relative to water- and CH-rich biological regions using Raman bands associated with DMSO, water, and lipid/protein content. A NoRI-inspired matrix decomposition approach was also explored to estimate relative contributions from DMSO, water, and biological tissue components, while accounting for limitations introduced by optical attenuation.
The resulting chemical maps provide spatial evidence for evaluating whether DMSO-d6 penetrates the chorion and whether it reaches internal embryonic tissue under the experimental soaking conditions. This work demonstrates the utility of confocal Raman microscopy combined with custom computational analysis for non-destructive, spatially resolved assessment of cryoprotectant transport in zebrafish embryos. The approach also highlights key challenges in quantitative Raman imaging of biological samples, including depth-dependent attenuation, baseline variability, and the need for chemically representative component models.