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Heptane and isooctane as embedding fluids for high‐pressure freezing of Petunia ovules followed by freeze‐substitution

In comparison with other fixation methods, high‐pressure freezing and freeze‐substitution of Petunia ovules lead to improved ultrastructural preservation of all tissues. Crucial for adequate high‐pressure freezing is the absence of air in the specimen sandwich; air has to be replaced by an embedding fluid. Frequently, 1‐hexadecene is used for this purpose. Using 1‐hexadecene as an embedding fluid resulted in only 5–10% of Petunia ovules being preserved without disturbance of the ultrastructure due to ice‐crystal damage. Since 1‐hexadecene is not soluble in acetone at − 90 °C, freeze‐substitution is hindered when ovules remained completely surrounded by it; this results in recrystallization when the temperature is raised. We tested and compared the suitability of heptane and isooctane as embedding fluids for high‐pressure freezing and freeze‐substitution, reasoning that because of their low melting points and low relative densities, phase separation during freeze‐substitution would result in complete exposure of the ovules to the substitution medium, leading to adequate freeze‐substitution. Using either heptane or isooctane as an embedding fluid yielded up to 90% ice‐crystal‐free ovules. Both compounds, however, have some damaging effects on the outer one or two cell layers of the ovule, but not on the inner tissues.