One of the challenges in using a dorsal skin flap chamber (DSFC) to study freeze injury in rodents is to achieve a controlled and well-characterized cryolesion within the ∼1 cm viewing area. A novel freezing device was designed that allowed optical access to the DSFC tissue, thus permitting the simultaneous use of various imaging methods (e.g. fluorescence, infrared) throughout a cryosurgery procedure. Stainless steel annuli were implemented with an internal 2.3 mm channel for circulation of cold nitrogen vapor. Inlet and outlet ports were manufactured from hypodermic needles. A machined external groove on the surface of these rings provided the point of attachment for interfacing with each side of the DSFC. Using thermocouples and numerical modeling, temperature gradients in the viewable tissue were estimated to be less than 1°C/mm and highly reproducible freezing rates between 3 and 9 °C/min were achieved in the tissue. At the fastest pump rates the apparatus was capable of producing temperatures as low as −110 °C on the inner surface of the chamber. When these rings were cooled to their lowest temperatures and placed in contact with the DSFC, tissue cooling rates of ∼75 °C/min could be achieved. Numerical modeling results demonstrated that the temperature gradient throughout the thickness of the tissue was minimal, suggesting that surface temperature measurements would be representative of the temperature throughout the thickness of the intravital preparation.

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