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.

冷冻保护剂对于确保生物组织在冷冻保存和随后的升温过程中能够在液氮暴露下存活至关重要。冷冻保护剂、细胞和茎尖之间的基本相互作用是一个尚未充分研究的领域，并且确切的保护机制仍然难以捉摸。通过将相干反斯托克斯拉曼散射显微镜与明场显微镜相结合，我们确定了广泛使用的冷冻保护剂二甲亚砜、乙二醇和甘油渗透活水稻（Oryza sativa）的速度。）愈伤组织细胞以及这种渗透如何与细胞反应相关。用明场显微镜观察到的一些细胞反应，例如质壁分离和去质壁分离，是对冷冻保护剂渗透的反应。本文报道的明场和相干反斯托克斯拉曼显微镜相结合的观察结果表明，冷冻保护剂渗透发生在质壁分离结束之前和去质壁分离结束之前。此外，冷冻保护剂渗透到没有明显质壁分离和去质壁分离的细胞中。总体而言，细胞响应时间比渗透时间长得多。