Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

Discussion:
Intrinsic and induced autofluorescence of formalin-fixed tissue samples cause well
known problems restricting the applicability of standard fluorescence microscopy
techniques. Overlapping of autofluorescence with the emission spectra of
fluorescently labeled targets seriously complicates detection, localization and
quantitation of specific signals, or provoke false-positive results and erroneous
interpretations. We addressed this issue and focused our studies on archival
specimen of human pancreatic tissue. Using formalin-fixed paraffin-embedded
pancreatic tissue samples, we first evaluated the localization and intensity of
autofluorescence. We observed a widespread distribution of autofluorescent
materials in these pancreatic specimens. As illustrated for other histological sections
before (Billinton and Knight, 2001), we also noticed severe autofluorescence within a
wide range of the visible light spectrum. The overall intensity of the detected
autofluorescence varied to a certain degree with the selected fluorescence emission
and excitation spectra. However, technical characteristics of the microscopy
equipment, such as the transmission quality of filter sets and optics, or the sensitivity
of the camera CCD, vary also significantly within the used wavelength ranges and
certainly have an impact on the recorded signal intensity. It is also noteworthy that no
change in intensity nor in signal quality of autofluorescence was observed before and
after removal of paraffin or retrieval of epitopes with the used histological sections
(data not shown). We identified distinct stromal and cellular components of formalinfixed
pancreatic tissue that exhibit high intensity of autofluorescence with certain
variations between different filter sets. Erythrocytes were shown to emit particular
bright fluorescence when Texas Red or Cy3 filter sets were used. Connective tissue
and fibers showed notably high autofluorescence signals with the DAPI filter set,
whereas pancreatic acini displayed a rather broad spectrum of intense
autofluorescence. As a result, the distribution of autofluorescence within a pancreatic
tissue section is not homogeneous. Certain tissue components, cell types, or even
subcellular compartments contribute peaks of bright fluorescence to a rather
moderately emitting surrounding area. We demonstrated that such pattern of
autofluorescence is capable to mimic or completely obscure the selective
immunostaining with fluorophores and thus provoke erroneous interpretation and
compromise the validity of fluorescent labeling approaches in immunohistochemistry.
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