Formalin Fixation and Cryosectioning Cause Only Minimal Changes in Shape or Size of Ocular Tissues

Advances in imaging have made it increasingly common to study soft tissues without first embedding them in plastic or paraffin and without using labels or stains. The process, however, usually still involves fixation and cryosectioning, which could deform the tissues. Our goal was to quantify the morphological changes of ocular tissues caused by formalin fixation and cryosectioning. From each of 6 porcine eyes, 4 regions were obtained: cornea, equatorial and posterior sclera, and posterior pole containing the optic nerve head. Samples were imaged using visible light microscopy fresh, 1-minute and 24-hours post-fixation, and post-cryosectioning. Effects were assessed by 14 parameters representing sample size and shape. Overall, formalin fixation and sectioning caused only minimal changes to the ocular tissues, with average percentage parameter differences of 0.1%, 1%, and 1.2% between fresh and post-fixing by 1 minute, 24 hours, and post-cryosectioning, respectively. Parameter changes were not directional, and were only weakly dependent on the duration of fixation and the region of the eye. These results demonstrate that formalin fixation and cryosectioning are good choices for studying ocular tissue morphology and structure, as they do not cause the large tissue shrinkage or distortions typically associated with other, more complicated, techniques.