Microscopic examination of an H&E section of kidney proves difficult; some areas cannot be brought into focus while others show excellent detail. This is most likely due to:

• A. Mounting medium on top of the cover glass
• B. Dirty objective lens
• C. Inadequate clearing
• D. Air bubbles in mounting medium

Answer: (A)

The focal mismatch across the slide comes from how the sample is mounted. If mounting medium sits on top of the cover glass rather than between the tissue and the coverslip, an air gap is created between the tissue and the glass. This adds an uneven optical path and variable thickness across the field, so some regions sit at the objective’s focal plane while others are out of focus. Proper mounting places the medium directly between the tissue and the coverslip to give a uniform refractive index and a flat, single focal plane. Dirty lenses, inadequate clearing, or air bubbles would produce different, more uniformly problematic effects across the field, rather than this variable focus pattern.