• photobleaching;
  • multiple parameters;
  • single fluorophore;
  • cell microarray system


The analysis of single cells with multiple parameters in flow cytometry or microscopy requires suitable combinations of fluorophores and optical filters. The growing demands for the multiplex analysis of cells increase the requirements for developing new fluorophores and techniques. We have developed a novel method of analyzing a large number of cells with multiple parameters on a single-cell basis using a single fluorophore. Cells were arrayed onto a microwell array chip with an array of 45,000 microwells, which could capture single cells, stained with a phycoerythrin (PE)-conjugated antibody to a marker, and analyzed with a cell-scanner. After analysis, we photobleached the PE molecules by irradiating the sample with blue light. Because the fluorescence of PE was not recovered after the photobleaching and the analyzed cells remained in the same microwells on the chip, we could repeatedly stain and analyze the same cells with other markers using PE. We applied a method of analyzing lymphocytes from 100 μL of peripheral blood for cytokine secretion and expression of intracellular proteins as well as for multiple cell surface markers. This novel method enables us to analyze multiple markers with a single fluorophore using a simple apparatus. The method may expand the scope of cytometry. © 2008 International Society for Advancement of Cytometry