There is a growing interest for flow and image cytometry analytes permitting the simultaneous discrimination of 6-10 fluorochromes. We have derived a strategy to optimize the factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. Following a spectrophotometric analysis of 14 fluorochromes conjugated to streptavidin (SA), a set of 7 spectrally separable SA- dyes and appropriate filter combinations were selected for evaluation in image cytometry. The SA-dyes were bound to latex particles labeled with biotinilated mIgG1 and the emissions of all fluorochromes detected by each filter combination were measured. The resulting crosstalk matrix served as the basic tool for final selection of dyes, design of optimal trichroic beamsplitter and filter combination, modulation of illumination and mathematical correction of residual spectral overlap. Using this strategy we demonstrated that latex-bound SA conjugates of Cascade Blue, Lucifer Yellow, FITC, R-PE, Red613, PerCP and APC could be discriminated. More recently we extended the applicability of the technique by analyzing blood cells bound to glass slides. The same field could be initially measured for autofluorescence and non-specific IgG binding and then remeasured for specific binding of lineage markers. The ability to use paired measurements of background and total fluorescence is a significant advantage of image over flow cytometry.

Date Published: 2 May 1997
PDF: 8 pages
Proc. SPIE 2982, Optical Diagnostics of Biological Fluids and Advanced Techniques in Analytical Cytology, (2 May 1997); doi: 10.1117/12.273637

Published in SPIE Proceedings Vol. 2982:
Optical Diagnostics of Biological Fluids and Advanced Techniques in Analytical Cytology
Robert C. Leif; Alexander V. Priezzhev; Toshimitsu Asakura; Robert C. Leif, Editor(s)