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The fundamental interactions between plant cells and cryoprotectants during vitrification are understudied in the field of plant cryopreservation. Within this area of research, real time cryoprotectant permeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice callus cells when in mixtures with other cryoprotectants. Specifically, we use coherent anti-Stokes Raman scattering (CARS) microscopy to observe the permeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibrational frequencies that correspond to C-D stretching modes of the cryoprotectant molecules. Additionally, we measure cell plasma membrane responses to PVS2 exposure using brightfield microscopy. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell plasma membrane responses observed and therefore permeation and cell plasma membrane response do not appear to be directly correlated. In addition, we observe that cryoprotectant permeation into plant cells occurs more quickly and more uniformly when cryoprotectants are in PVS2 solution than when they are in single component aqueous solutions., (Copyright © 2024 Society for Cryobiology. All rights reserved.)

Cryopreservation has emerged as a low-maintenance, cost-effective solution for the long-term preservation of vegetatively propagated crops. Shoot tip cryopreservation often makes use of vitrification methods that employ highly concentrated mixtures of cryoprotecting agents; however, little is understood as to how these cryoprotecting agents protect cells and tissues from freezing. In this study, we use coherent anti-Stokes Raman scattering microscopy to directly visualize where dimethyl sulfoxide (DMSO) localizes within Mentha × piperita shoot tips. We find that DMSO fully penetrates the shoot tip tissue within 10 min of exposure. Variations in signal intensities across images suggest that DMSO may interact with cellular components, leading to its accumulation in specific regions.

Cryoprotectants allow cells to be frozen in liquid nitrogen and cryopreserved for years by minimizing the damage that occurs in cooling and warming processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not entirely evident. This contributes to the arduous process of optimizing cryoprotectant formulations for each new cell line or species that is conserved. Coherent anti-Stokes Raman scattering microscopy facilitates the visualization of deuterated cryoprotectants within living cells. Using this technique, we directly imaged the location of fully deuterated dimethyl sulfoxide (d 6 -DMSO), the deuterated form of a commonly used cryoprotectant, DMSO, within rice suspension cells. This work showed that d 6 -DMSO does not uniformly distribute throughout the cells, rather it enters the cell and sequesters within organelles, changing our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration within different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distributions of these molecules change when different cryoprotectants are used.