2.3. Microscopic Analysis

From the frozen stored snakehead block (−20 °C), two pieces (approximately 5 mm in length) were cut from the center, transversally to the muscle fiber, using a blade previously cooled to −20 °C. An indirect method, known as isothermal freezing substitution, was used to observe spaces produced by ice crystals in the fish tissue, similar to that reported in a previous study of ice crystal formation in salmon [18]. The frozen snakehead blocks were embedded with an embedding agent (O.C.T. Compound, Japan), placed in a low temperature environment until the embedding agent had solidified, and then sliced into sections. Using a semi-conductor frozen slicer (Cryostar N × 50, Thermo Electron Corp., USA), the embedded samples were sliced into 7-μm-thick specimens along the direction of the muscle fibers, and hematoxylin-eosin staining was used to observe ice crystals.

All prepared slides were observed using a light microscope (Olympus B × 41, Japan) equipped with a camera (Olympus Corp., Japan). Images of the slides were treated using Image J software (National Institutes of Health, USA). Three parameters, namely the cross-sectional area, equivalent diameter, and roundness, were used for microscopic analysis. The roundness (R) was calculated as follows:

where A is the cross-sectional area and P is the perimeter (the length of the outside boundary of the observed object). For each case, more than 100 frames (ice crystals or muscle fibers) were evaluated.