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A comparison of the detection sensitivity of lymphocyte membrane antigens using fluorescein and phosphor
H B Beverloo1, A van Schadewijk, H J Zijlmans
1Department of Cytochemistry and Cytometry, Sylvius Laboratory, University of Leiden, The Netherlands.
Insights
Time-resolved fluorescent dyes offer superior sensitivity for immunocytochemistry compared to conventional fluorescent dyes like FITC. This enhanced sensitivity is crucial for accurately detecting specific cellular targets, such as the CD4 epitope on human lymphocytes.
Area of Science:
- Immunocytochemistry
- Fluorescence Microscopy
- Cell Biology
Background:
- Immunocytochemical staining is vital for identifying cellular markers.
- Conventional fluorescent dyes like FITC have limitations in sensitivity.
- Time-resolved fluorescence microscopy offers potential improvements.
Purpose of the Study:
- To compare the sensitivity of conventional (FITC) and time-resolved fluorescent dyes (immunophosphors) in immunocytochemistry.
- To evaluate the performance of these dyes using both conventional and time-resolved fluorescence microscopy.
- To assess the detection of the CD4 epitope on human lymphocytes.
Main Methods:
- Paraformaldehyde-fixed human lymphocytes were stained for the CD4 epitope.
- Indirect immunofluorescence was performed using FITC and time-resolved luminescent immunophosphors.
- Cells were examined visually and quantitatively using conventional and time-resolved fluorescence microscopy with a CCD camera.
- A double-blind study design was employed for visual assessment.
Main Results:
- Conventional fluorescence microscopy showed better sensitivity with phosphor conjugates over FITC.
- Quantitative assessment with time-resolved microscopy further highlighted the superior sensitivity of phosphor conjugates.
- Time-resolved microscopy effectively suppressed background fluorescence, achieving a suppression ratio greater than 1 to 10^6.
Conclusions:
- Time-resolved luminescent immunophosphors demonstrate enhanced sensitivity in immunocytochemical staining compared to FITC.
- Time-resolved fluorescence microscopy provides superior signal-to-noise ratio by suppressing autofluorescence and background.
- These findings support the use of time-resolved immunophosphors and microscopy for more sensitive cellular analysis.
Abstract:
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).