Cytofluorometry : technique and applications

Abstract: The aims of this investigation were to examine the conditions for rapid fluorescence measurements on cellular material, to improve the performance of measurement systems for microscope fluorometry, and to apply this technique in studies on mast cell biology. Fluorescence fading, a general complication of cytofluorometry, was studied during illumination times in the millisecond range, and a new fading phenomenon characterized by short duration and rapid recovery was described. The findings of the study formed the basis for the construction of two instfurnent systems for microscope fluorometry based on Leitz MPV I and MPV II microscope photometers. Rapid fluorescence measurements were performed by a completely automatic measuring sequence, except forselectionandfocusing of the objects to be measured. Automation was mainly achieved by the integration of computers in the measurement systems, which also resulted in easily interchangeable programdetermined measuring routines and proper data processing and presentation of results. The systems were mainly used for rapid analysis of cell populations. The precision of the measurements was improved by different standardization techniques, and the measuring speed, about 500 cells per h on well prepared specimens, was high enough to permit analysis of relatively large cell populations within a reasonable time.The cytofluorometric technique was applied to studies on the biology of the connective tissue mast cell. Rat peritoneal mast cells were used for this purpose. The proliferation of mast cells was estimated by cytofluorometric measurements of DNA in mast cell populations after staining with the fluorescent dye Hoechst 33258. Formaldehyde-induced fluorescence was used to study the uptake and turnover of dopamine in mast cells in vivo. Measurements of the mast cell content of heparin, a constituent of the mast cell granule matrix, were performed by a combination of microscope fluorometry, which permits visual identification of the cells, and flow cytofluorometry, by which rapid measurements of large populations can be made.