Fluorescence-aided detection of microdamage in compact bone

Abstract

En bloc staining with basic fuchsin is an established method for demonstrating microdamage in bone. Using transmitted light microscopy, variations in light intensity, depth of focus and magnification are necessary to distinguish fully-stained microcracks generated in vivo, from partially-stained or unstained artefactual cracks due to cutting and machining. This process is both difficult and time-consuming. In this study, 2 methods were used to examine fuchsin-stained microcracks in human rib sections, transmitted light and epifluorescence microscopy. No differences were found in crack number, density or length between the 2 methods indicating comparable accuracy. Using green epifluorescence, only microcracks containing fuchsin fluoresced orange against the darkfield background, enabling unstained, artefactual cracks to be screened out. Under UV epifluorescence, microcracks stained through the full 100 μm depth of the section fluoresced purple. Partially-stained artefactual cracks failed to fluoresce and were screened out. Epifluorescence is a simple, rapid and accurate screening method for differentiating fully-stained from artefactual microcracks in bone.