Automated data acquisition by confocal laser scanning microscopy and image analysis of triple stained immunofluorescent leukocytes in tissue.

The aim of the study was to develop a novel system permitting automated analysis of multicolor immunofluorescence-staining of cells in solid tissues which would be comparable to the analytical capacity of flow cytometry. In the user friendly automated data acquisition and image processing system which was established, the software includes a set of pre-defined processing steps for improved object identification and can be either interactive or fully automatic. As with multi color flow cytometry, stained cells can be analyzed in a fully automated manner regardless of tissue type. The software organizes computerized sample movement, autofocus, laser readjustment, data capture and storage as well as calculation. Data are presented as histograms indicating the staining intensity and frequency of each antigen, and in dotplots with each channel plotted against the other. The calculated statistics give information about how many of the cells are single-, double- or triple-reactive and how intensely they react with the respective antibodies. Comparison of data generated by this automated fluorescence confocal laser scanning microscopy (AF-CLSM) with flow cytometry using triple stained peripheral blood lymphocytes revealed a highly significant correlation between the methods (P<0.001). A correlation was also observed when sections triple stained for anti-CD45, -CD3 and -CD8 were analyzed by AF-CLSM and the data were compared to visual/manual cell counting (P<0.01). The AF-CLSM system permits for the first time, fast (online) and reproducible analysis of immunofluorescence staining of an unlimited number of cells in tissue sections. The software, including a manual, is available for a small fee to cover the costs of printing and postage.