Widefield microscopy provides high quality multi-color fluorescence imaging comparable to confocal microscopy but in a more time efficient and cost effective manner. A motorized stage and integrated software allow multi-color imaging of up to three stains on fixed bovine cells in less time than a confocal. Quantitative analysis tools within the software allow measurement of fluorescence intensity, distribution and 3D reconstruction from z-stack images. Widefield microscopy is thus a suitable alternative to confocal microscopy for quantitative fluorescence applications.