|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥1,900,000 (Direct Cost : ¥1,900,000)
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
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