Confocal microscopy of multiple stained biological specimens using fluorescence lifetimes

摘要

We here report on using fluorescence life times recordings in confocal microscopy to detect individual fluorophores in multiple stained tissue. Using indirect fluorescence immunohistochemistry with secondary antisera conjugated to the fluorophores Lissamine Rhodamine (LRSC), Texas Red or Cyanine 3.18 (Cy-3); one, two or three epitopes were labelled in tissues from rat spinal cord and dorsal root ganglia. The tissue sections were examined and analyzed in a beam-scanning confocal microscope equipped with devices for fluorescence lifetime measurements. The results show that fluorophore life-times can be used to separate two or more fluorophores in individual axon terminals, provided that the fluorophore labelling is strong enough and the life-times of the deployed compounds are sufficiently separate. Thus, the presence of Cy-3, LRSC and Texas Red, as well as mixtures of these compounds could be detected in individual tissue profiles. Contribution by tissue autofluorescence was unmistakably identified by its complex multiexponential emission decay. We also show that fluorescence lifetimes differs between antiserum-conjugates for one and the same fluorophore, and the possibility to use fluorophore life-times to probe chemical environmental changes in situ is discussed. The implementation of a life-time recording device in a confocal microscope has the advantage of providing a good spatial resolution. Furthermore, by deploying fluorophores emitting within the same wavelength band, problems due to chromatic errors will be completely avoided.