In this study we compared the sensitivity of immunocytochemical procedures, using conventional and time-resolved fluorescent dyes, in a model system consisting of paraformaldehyde-fixed human lymphocytes. The lymphocytes were stained for the presence of the CD4 epitope by indirect immunofluorescence using FITC as label or by using time-resolved luminescent immunophosphors. These immunophosphors were primarily developed for use under time-resolved fluorescence conditions, but they are also very well suited for use in conventional fluorescence microscopes. The differently labeled cells were first examined visually with a conventional fluorescence microscope in a double-blind study. The fluorescence was also measured with a CCD camera mounted on a specially constructed time-resolved fluorescence microscope which allows the suppression of the fast decaying fluorescence, thereby permitting visualization of the specific, slowing decaying luminescence of the phosphor label. With this microscope FITC and immunophosphor labeled lymphocytes were compared under normal conditions (i.e., continuous excitation) and under conditions of time-resolved registration. Conventional fluorescence microscopy revealed a better sensitivity in favor of the phosphor conjugates. This difference became more prominent when the preparations were quantitatively assessed with the CCD-time-resolved microscope. Time-resolved microscopy permitted a suppression of fast decaying fluorescence better than 1 to 10(6).
Novel Time-Resolved Fluorescence Microscope System Using TCSPC and Multi-Frequency Techniques
