Direct observation of common cryoprotectant permeation into rice callus by CARS microscopy

Cryoprotectants are vital to ensuring that biological tissue can survive liquid nitrogen exposure during cryopreservation and subsequent warming. The fundamental interaction between cryoprotectants, cells, and shoot tips is an understudied area, and the exact mechanism for protection remains elusive. By coupling coherent anti-Stokes Raman scattering microscopy with bright-field microscopy, we determine how quickly the widely used cryoprotectants dimethyl sulfoxide, ethylene glycol, and glycerol permeate living rice (Oryza sativa) callus cells and how this permeation correlates with cellular responses. Some cellular responses observed with bright-field microscopy, such as plasmolysis and deplasmolysis, occur in response to cryoprotectant permeation. The observational results from the combination of bright-field and coherent anti-Stokes Raman microscopy reported here show that cryoprotectant permeation occurs before the end of plasmolysis and well before the end of deplasmolysis. Further, cryoprotectants permeate cells where there is no apparent plasmolysis and deplasmolysis. Overall, cell response times are substantially longer than permeation times. [Display omitted] •Cryoprotectants permeate cells more quickly than cellular responses indicate•Some cells do not respond to cryoprotectant permeation•Coherent Raman shows the real-time distribution of molecules in live cells Using coherent Raman microscopy, Samuels et al. directly image the real-time permeation of common cryoprotectants into live plant cells. Generally, permeation time is assumed based on cell responses, but these results indicate that the beginning of permeation occurs before the cell responses commonly attributed to cryoprotectant permeation.