Chemical cytometry uses capillary microseparation with highly sensitive detection for detailed chemical analyses of single cells. Here, we studied microscopy and chemical cytometry in measurements of total fluorescence from single cells. An inverted fluorescence microscope was modified so that the fluorescence intensity of a sample could be automatically measured for different vertical positions of the sample with respect to the objective lens. The capillary of a custom-made chemical cytometer was mounted in a vertical position over the microscope stage. A diluted suspension of 4T1 (mouse mammary gland tumor) cells stably expressing green fluorescent protein (GFP) was placed on a microscope slide. A single cell was positioned in the center of the field of view of the microscope. The intensity of its total GFP fluorescence was measured with the microscope detector first. The cell was then analyzed in the chemical cytometer as follows. The cell was injected into a capillary and lysed to form a homogeneous cellular lysate. The lysate was driven through the capillary by pressure and its GFP fluorescence was quantified at the output of the capillary with a laserinduced fluorescence (LIF) detector. We demonstrated for the first time that in microscopy, the maximum fluorescence signal, as well as maximum signal to noise ratio, could be obtained when the cell was in one of two extremely out-of-focus positions. We proved that in chemical cytometry, the intensity of fluorescence had no memory of cell geometry and depended solely on the amount of GFP. This feature ofchemical cytometry allowed us to use this technique as a reference method in the study and optimization of quantitative fluorescence microscopy. Using chemical cytometry as a reference method and 4T1 cells as a biological model, we proved that microscopy can be employed for reliable measurements of total fluorescence intensity from single cells in a heterogeneous population of non-synchronized cells. This finding opens the opportunity for a wide use of tandem microscopy/chemical cytometry in analytical cytology.

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