Stimulated Raman Scattering (SRS) microscopy enables visualization and quantification of cryoprotective agent (CPA) concentration in Daphnia magna

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 often takes years. In this study, we present a Stimulated Raman Scattering (SRS) microscopy-based method for the spatiotemporal quantification of dimethyl sulfoxide (DMSO-d6) uptake in living Daphnia magna. DMSO-d6 uptake in D. magna was found to be non-uniform and anatomically dependent, with similar uptake levels in the head and heart that were substantially higher than those in the carapace. Furthermore, we observed that internal DMSO-d6 concentrations converged to similar levels at their respective LT50 times, regardless of the incubation concentration. This approach enables in vivo visualization and measurement of CPA distribution across a whole organism. Our findings lay the groundwork for developing SRS-guided cryopreservation strategies in D. magna, with potential translational relevance to other complex biological systems.