Optimizing multiple fluorochrome bone histodynamic markers

Abstract

In vivo fluorochromes that adsorb to bone mineral, principally tetracyclines and their derivatives are well established histologic markers for assessing the mineralizing surfaces of bone (5). To distinguish clearly which surfaces are adsorbing fluorochrome at a particular time, fluorochromes of sufficiently different emission spectra to appear as different colours have been administered at different times prior to histologic examination (3,6,10,11,13,14, 17,18,19). For example, Parfitt and colleagues examined human bone biopsies after administration of Oxytetracycline (green label) and Demethylchlortetracycline (yellow label) (13). To consider the possibility of inhibition effects in some patients, Oxytetracycline was administered first while in others the administration order was reversed. These authors found that the Oxytetracycline label was shorter than the length of the Demethylchlortetracycline label regardless of the order in which the labels were administered. Both labels were measured under the same fluorescent conditions. In experimental animal studies, methods have been developed employing three fluorochromes each with emission spectra which appear as different colours. Most investigators examined all labels under the same fluorescent conditions (10,11,17). Commonly, the investigators omitted to specify precisely the light source, optics, and in particular the excitation and emission spectra of the fluorescent filters used for examination (3,6,19). A pioneering exception is the work of Solheim who demonstrated using transmitted light fluorescent microscopy that the fluorescent intensity of Tetracycline, Alizarin and other markers varied with the excitation and barrier filter used (18). Pinholt and Kwon using three fluorochromes, and two filter blocks noted the detection of labels but did not measure mineralizing surfaces (14) D