Stepwise Immunofluorescence Computed Tomography (ICT) protocol coupled with non-linear optical microscopy.

<p>(A) Serial section BMMA embedded tissue block (50 nm–5 µm). (B) SHG imaging visualises fibrillar collagen. (C) Immunofluorescent labelling and subsequent imaging. (D) Strip antibodies with glycine hydrochloride (pH 2.5) accordingly and re-stain to visualise multiple antigens in the same tissue sections. (E and F) Overlay images or repeat iterative antibody labelling. (G) Align and reconstruct. (H) Quantitative and qualitative analysis of cell populations may then be performed from 3-D reconstructions. (Red arrow: Meibomian gland segmented volume, Green arrow: CK1, Blue arrow: collagen SHG signal orthoslice).