Quantitative light microscopy of combined perfusion and freezing processes

The rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1-5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryomicroscopy over a range in excess of 50 to -100 degrees C. A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and -10 degrees C. The technique involves mounting a single islet onto the low-temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.