Three-Dimensional Microstructure of Transport Phenomema during Freezing if Biolo cal Materials

生物材料的冻结是低温外科和低温保存中最基本的现象。研究冰晶和细胞在冷冻过程中的微观行为，对于研究细胞的冷冻损伤机制和冷冻保护剂对细胞的保护具有重要意义。然而，生物材料的冻结和解冻通常是在三维(3D)中瞬时和空间地进行的。因此，需要进行实时的三维观察，以了解加工过程中微观组织的细节。这种观察永远不能用光学和电子显微镜进行，因为这些显微镜只能产生沿样品厚度方向平均的2D图像。此外，所需的样品固定不允许观察相同的样品。最近，研制了一种激光共聚焦扫描显微镜。这是一种非侵入性的方法，无需f…即可产生生物材料的光学层析图像。对样品进行更多的固定和切片。CLSM可以生成3D图像。利用CLSM和荧光染料，实时显示了生物材料冻结和解冻过程中冰晶和细胞的3D行为。研究了冰晶的形态以及冰晶与细胞之间的相互作用。吖啶橙作为一种荧光染料，其最大激发波长为492 nm，分别产生绿色和红色荧光，最大发射波长分别为530am和640 nm。通过它的染色，可以用不同的颜色区分冰晶、未冷冻的溶液和细胞。生物材料为人红细胞悬液和新鲜鸡肉(胸肌)。在生理盐水(0.154M氯化钠)(PS)和生理盐水(PS+甘油)中，人红细胞悬液在细胞外冷冻过程中，所有平面和细胞固液界面以及树枝状界面的微观结构均为3D。后者中的冰液界面呈波浪形，很容易与红细胞的形态相一致。这与前者界面的单调曲线轮廓形成了对比。在四种降温速率和升温速率组合下，冻融组织的微观结构也是三维的。在缓慢降温过程中，细胞外发生冻结，相邻的肌纤维被撕裂。解冻后，肌纤维之间仍有不可逆转的裂缝。相反，细胞外和细胞内的冻结发生在快速降温过程中，解冻后不仅肌纤维之间仍有裂缝，而且纤维内部也有许多细小的裂缝。较慢的升温促进了纤维的变形。在组织中添加2.0M二甲基亚砜可以减少冰块的数量、裂缝的大小、解冻后肌纤维的变形等。

Freezing of biological materials is a most fundamental phenomenon in cryosurgery and cryopreservation. Investigation of microscopic behavior of ice crystals and cells during the freezing is of great importance in relation to the mechanisms of freezing-injuries of cells and protection of cells due to cryoprotectants. However, the freezing and thawing of biological materials generally proceeds transiently and spatially in three-dimensions(3D). Therefore, 3D observation in real time is required to understand the details of microstructure in the process. Such observation could never be conducted using optical and electron microscopes because these microscopes produce only 2D images averaged in the direction of thickness of the sample. Furthermore, the required fixation of the sample does not allow observation of the same sample. Recently, a confocal laser scanning microscope (CLSM) was developed. This is a noninvasive method that produces optical tomograms of biological materials without f … More ixation and slicing of a sample. The CLSM can produce 3D images.3D behavior of ice crystals and cells during the freezing and thawing of biological materials was visualized in 3D in real time using a CLSM and a fluorescent dye. Investigated were the morphology of ice crystals and the interaction between ice crystals and cells. Acridine orange as a fluorescent dye has maximum excitation wavelength of 492nm and generates green and red fluorescences, respectively, with emission maxima at 530Am and 640nm. The stain by it enabled ice crystal, unfrozen solution, and cells to be distinguished with different colors. Biological materials were human red blood cell suspensions and fresh white meat of chicken (pectoral muscles).The microstructure near the freezing interface during the extracellular-freezing of human red blood cell suspensions was 3D for all flat and cellular solid-liquid interfaces in physiological saline (0.154M NaCl) (PS) and for a dendritic interface in physiological saline with 2.4M glycerol (PS+Gly). The ice-solution interface in the latter is wavy and easily conforms to the morphology of the red blood cells. This contrasts with the monotonically curved outline for the interface in the former.The microstructure of the tissues through the freezing and thawing was also 3D for four combinations of cooling-rate and warming-rate. In the slow-cooling, the extracellular-freezing happened, and adjoining muscle-fibers were tore. Irreversible cracks remained behind between the muscle fibers after the thawing. In contrast, the extracellular- and intracellular-freezing happened in the rapid-cooling, After the thawing, the cracks remained behind not only between the muscle fibers but also many small cracks remained behind in the fibers. The slower-warming promoted the deformation of the fibers. Addition of 2.0M dimethyl sulfoxide in tissues decreased the amount of ice, the size of cracks, the deformation of the muscle fibers after the thawing, etc. Less