New advances in fluorochrome sequential labelling of teeth using seven different fluorochromes and spectral image analysis

Abstract

Fluorochrome sequential labelling of mineralizing tissues is commonly used in different fields of clinical and basic research. Recently we improved polychrome fluorescent sequential labelling of bone by applying spectral image analysis to discriminate seven different fluorochromes. Although basic mineralization processes of bone and teeth follow comparable principles, the respective tissues differ in terms of matrix composition and mineral assembly. The aim of this study therefore was to investigate the feasibility of this new technique for polychrome sequential labelling of teeth and to demonstrate the advantages in the field of dentistry. Furthermore, the exact labelled area of each fluorochrome could be measured, even in regions of overlapping fluorochromes. The technique presented may provide a basis for further investigations of mineralization processes of different anatomical dental structures.

Fig. 1

Region of rat mandibular bone with a lower right incisor. (a) Haematoxylin–eosin staining and (b) fluorescence image using a filter set for green and red fluorescence (SKY, ASI, Israel) (scale bar = 1 mm). The area marked in the rectangle is magnified in (c) and (d). Large arrows mark a calcifying dentine microstructure (denticle) magnified in panels (e)–(k). (c,d) Conventional fluorescence images showing unlabelled enamel and the sequentially labelled dentine using (c) a triple band filter (SKY, ASI) and (d) a long-pass emission filter (no. 01, Zeiss). (e,f) A calcifying dentine microstructure recorded by conventional fluorescence imaging using the same filter sets as in (c) and (d). (g) Identical area recorded by spectral image acquisition using the long-pass emission filter. Note that seven different fluorescent bands are visible simultaneously. In contrast to the conventional images, the hematoporphyrin band was clearly visible by spectral image acquisition. (h–k) Linear unmixing enabled single depiction of each fluorochrome, allowing exact quantification of the labelled area: (h) doxycycline/rolitetracycline (note that the width of the band is caused by two fluorochromes that were not distinguishable), (i) calcein and (k) xylenol orange. Abbreviations: cb, calcein blue; x, xylenol orange; c, calcein; ac, alizarine complexone; d/r, doxycycline/rolitetracycline; hp, hematoporphyrin; B, BAPTA. Scale bars in (c)–(k) = 50 µm.