Multiple Beam Interference Confocal Microscopy: Tool for Morphological Investigation of a Living Spermatozoon

The interference pattern obtained from a multiple internal reflection of a spermatozoon, sandwiched between the glass plate and the cover plate, was focused on the objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originating from the interference pattern and the optical sectioning of the confocal scanning system, enhanced the resolution and contrast in an impressive manner. These features permitted unprecedented images of the spermatozoon to be obtained at 0.1 µm optical sectioning and reconstruction of three-dimensional (3-D) images. With the help of optical sectioning, it was possible to estimate the thickness and the morphological details of the spermatozoon.