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Figure 1. The rate at which the extracellular concentration is changing as a function of time during three treatments. Linear change of temperature with time (– – –); `controlled concentration' (——); linear change in ice fraction with time (- - - - -).
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Figure 2. Cell recovery (normalized %) as a function of the proportion of the time that the rate at which the extracellular concentration is changing is decreasing. Experiment #1 (——); experiment #3 (- - - - -).
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Figure 3. Freeze-fracture electron microscopy of sperm suspensions cryopreserved within conventional straws (0.25 ml capacity). (a) Cross-fracture of whole straw—low magnification. (b) Detail of the etched freeze-concentrated glycerol. (c) Sperm tails (arrows) extending from freeze-concentrated matrix into the space previously occupied by an ice crystal. (d) Detail of a sperm bridging an ice crystal void. (e) Cell at the interface between ice and the freeze-concentrated matrix. (f) Distorted sperm cell.
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Figure 4. Light microscopy of thin-sectioned freeze-substituted straws. (a) Straw solidified to achieve constant ice fraction with time, a sperm tail bridging two freeze-concentrated zones is apparent (arrow); also, sperm tails extending from the freeze-concentrated matrix (arrowhead). (b) Straw solidified by the `controlled concentration' method, areas of granularity within the freeze-concentrated matrix are arrowed. Scale bar = 1 µm.
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Figure 5. Transmission electron microscopy of a thin-sectioned freeze-substituted straw solidified by the `controlled-concentration' method; a zone of low electron density around the cell is arrowed. Substituted ice crystals in the freeze-concentrated matrix are also apparent (arrowheads). Scale bar = 1 µm.
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Figure 6. (a) Estimated local undercooling as a function of time–water movement only. (b) Estimated local undercooling as a function of time—dominated by glycerol movement. `Controlled concentration' (——); linear ice (- - - - -); linear rate of temperature change (– – –).
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Figure 7. (a) Estimated mass transport as a function of time–water movement only. (b) Estimated mass transport as a function of time—dominated by glycerol movement. Controlled concentration (——); linear ice (- - - - -); linear rate of temperature change (– – –).
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