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Recognition of basic fuchsin prestained microfissures of intravital origin with fluorescence microscopy: validation
T Frisch1, M S Sørensen, P Bretlau
1Otopathological Laboratory, Department of Otolaryngology, Head and Neck Surgery F-2071, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark. tfrisch@RH.DK
Abstract:
For 70 years it has been suspected that not all microfissures in histological 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 artifactual microfissures in the load-bearing skeleton. Recently the presence of intravital microfissures has also been ascertained in temporal bones by these techniques. Due to the fluorescent properties of basic fuchsin it is possible to use epifluorescence microscopy for analysis of microfissures after bulk staining with basic fuchsin. This provides a more steady microscopic background and a sharper delineation of surface level structures since no projection from lower levels interfere. Artifactual cracks, which in transmitted light microscopy may look like darkly stained intravital microfissures due to refraction phenomena, become invisible or colorless. Epifluorescence microscopy enhances the detection of both smaller and larger prestained intravital microfissures, and leaves only a minor part of the cracks without certain categorization. The epifluorescence mode of analysis has the further advantage of being independent of slice thickness, making feasible whole-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 numerically 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 fistulae.
Insights
Repetitive stress may cause intravital microfissures in bone, not just artifacts. Epifluorescence microscopy effectively detects these bone microcracks in temporal bones, aiding otosclerosis research.
Area of Science:
- Histopathology
- Microscopy
- Skeletal Biology
Background:
- For 70 years, the origin of bone microfissures (artifacts vs. in vivo stress) has been debated.
- Undecalcified bone and bulk staining techniques differentiate artifactual from intravital microfissures in load-bearing bones.
- Intravital microfissures have recently been confirmed in temporal bones using these methods.
Purpose of the Study:
- To evaluate epifluorescence microscopy for detecting and analyzing intravital microfissures in human temporal bones.
- To compare epifluorescence microscopy findings with traditional light microscopy for microfissure analysis.
- To establish epifluorescence microscopy as a reliable method for studying bone microcracks.
Main Methods:
- Bulk staining of human temporal bone sections with basic fuchsin.
- Analysis of microfissures using epifluorescence microscopy.
- Comparison of epifluorescence microscopy with transmitted light microscopy.
- Serial stepwise grinding for whole-specimen analysis.
Main Results:
- Epifluorescence microscopy clearly distinguishes artifactual cracks from intravital microfissures.
- Artifactual cracks appear invisible or colorless under epifluorescence microscopy, unlike in light microscopy.
- Epifluorescence microscopy enhances the detection of both small and large intravital microfissures.
- Quantitative analysis (number and length) of microfissures in the otic capsule using epifluorescence microscopy matched light microscopy findings.
Conclusions:
- Epifluorescence microscopy is a reliable and effective method for analyzing intravital microfissures in temporal bones.
- This technique enhances microfissure detection and differentiation from artifacts.
- Epifluorescence microscopy offers advantages like a steady background and independence from slice thickness.
- The method holds significant potential for research into otosclerosis and perilymphatic fistulae etiology.