Sequential Photobleaching for increasing the Measurable Fluorochromes by Laser Scanning Cytometry

摘要

For immunophenotypic analysis more measurable parameters for the discrimination of leukocyte subsets are necessary. With a single scan six fluorochromes can be distinguished with the Laser Scanning Cytometer (LSC). Due to the number of PMTs the amount of simultaneously measurable fluorescences per scan is limited. Nevertheless, the amount of measurable colors can be improved to eight by appropriate change of the filter settings and two scans per specimen . Aim of this study was to use the special features of Slide based Cytometry (SBC) beyond filter change, remeasurement and merging to distinguish fluorochromes with similar emission spectra. The photosensitivity of fluorochromes that are excited and emit in a similar wavelength range may be very different. The number of measurable parameters per PMT was increased using photosensitivity of different fluorochromes as additional criteria. Peripheral blood leukocytes wer stained with antibodies conjugated to the fluorochromes APC, APC-Cy5.5 and Alexa-Fluor 633 and mounted on conventional uncoated glass slides with Fluorescence mounting medium. Specimens were excited in the LSC with th HeNe (633nm) Laser and measured at different filter settings (670/20nm-filter for APC/ALEXA633 and 710/20nm-filter for APC-Cy5.5). At this point, APC-Cy5.5 and APC/ALEXA633 were already distinguishable. In order to differentiate between APC and ALEXA633 photobleaching was performed by repeated excitation with the laser at 633nm. Control measurements proved that APC is much more sensitive against laser excitation, i.e. looses much more fluorescence intensity than ALEXA633. The separate measurements (before/after filter change and before/after bleaching) were merged into one file. The photostability of Alexa-Fluor 633 (1.02% bleach per scan) and APC (5.74% bleach per scan) are substantially different. Therefore, after bleaching and merging both fluorochromes can be distinguished and are regarded by the software as separate parameters. The fluorochromes APC/ALEXA633 and APC-Cy5.5 can be discriminated by changing the emission filters before bleach. By sequential photobleaching, change of filters and subsequent merging of the data the number of simultaneously measurable "colors" is substantially increased.