Recognition of basic fuchsin prestained microfissures of intravital originwith fluorescence microscopy: validation of a shortcut

For 70 years it has been suspected that not all microfissures in histologic al bone sections are artifacts, but that some are provoked in vivo through repetitive stress. The development of undecalcified bone techniques and of the bulk staining technique has established a method for demonstrating the existence of intravital cracks and enhanced the discrimination towards arti factual microfissures in the load-bearing skeleton. Recently the presence o f intravital microfissures has also been ascertained in temporal bones by t hese techniques. Due to the fluorescent properties of basic fuchsin it is p ossible to use epifluorescence microscopy for analysis of microfissures aft er bulk staining with basic fuchsin. This provides a more steady microscopi c background and a sharper delineation of surface level structures since no projection from lower levels interfere. Artifactual cracks, which in trans mitted light microscopy may look like darkly stained intravital microfissur es due to refraction phenomena, be come invisible or colorless. Epifluoresc ence microscopy enhances the detection of both smaller and larger pre-stain ed intravital microfissures, and leaves only a minor part of the cracks wit hout certain categorization. The epifluorescence mode of analysis has the f urther advantage of being independent of slice thickness, making feasible w hole-specimen analysis by serial stepwise grinding. The present study shows that the number and the length of microfissures in the human otic capsule, counted and measured under the epifluorescence microscope, equals numerica lly the findings in light microscopy enabling the routine use of this mode of analysis. This may prove to be of particular value in the research into the etiology and pathogenesis of otosclerosis as well as perilymphatic fist ulae.