Stimulated Raman Scattering (SRS) Microscopy Enables Visualization and Quantification of Cryoprotective Agent Concentration in <i>Daphnia magna</i>

Cryopreservation enables long-term storage and transport of biological specimens by minimizing or avoiding ice formation at low temperatures through the use of cryoprotective agents (CPAs). However, CPAs are often toxic at effective concentrations, necessitating precise optimization of exposure parameters such as concentration, incubation time, and temperature. Currently, the field lacks tools to monitor CPA diffusion and distribution in real time, especially in complex organisms, leading to iterative optimization that can take months to years. In this study, we present a Stimulated Raman Scattering (SRS) microscopy-based method for the spatiotemporal quantification of deuterated dimethyl sulfoxide (DMSO-<i>d</i>₆) uptake in living <i>Daphnia magna</i>. <i>D. magna</i> is a widely used model organism with favorable optical properties for noninvasive imaging and particular relevance to cryopreservation studies. DMSO-<i>d</i>₆ uptake in <i>D. magna</i> was found to be nonuniform and anatomically dependent, with higher concentrations measured in the head and heart relative to the carapace. Furthermore, we observed that internal DMSO-<i>d</i>₆ concentrations converged to similar levels at their respective median lethal time (LT₅₀), regardless of the incubation concentration. This approach enables <i>in vivo</i> visualization and measurement of CPA distribution across a whole organism. Our findings lay the groundwork for developing SRS-guided cryopreservation strategies in <i>D. magna</i>, with potential translational relevance to other complex biological systems.