Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. used to discriminate calcified bone and elastic fibers using the fluorescence microscope (Bradbeer et al. 1994). The selective fluorescence exhibited by these two main tissue components is based on the very faint eosin staining, while intensely eosinophilic substrates and those stained with the highly absorbing basic dyes show no fluorescence or just a very faint signal. Considering the possibility of using eosin fluorescence for the selective identification of some tissue components, we have extended this approach to the study of elastic fibers in hematoxylin-eosin (H&E) preparations. This paper then describes the results of observations with the fluorescence microscope of some H&E stained sections and evaluates the use of the confocal laser scanning microscope to image elastic fibers in the same preparations. Furthermore, we have also studied absorption and fluorescence spectra of eosin and phloxine, a closely related dye, in solution, to correlate them with the fluorescence detected in tissue sections.