Imaging fluorescently-labeled DNA in live cells with nanoscale precision shows significant promise as a diagnostic tool; however, the intrinsically stochastic nature of biological systems limits our ability to interpret meaningful signals from the noise. Balancing the constraints of high-resolution microscopy while attaining the necessary number of samples for statistical significance means expensive and slow measurements. Imaging-flow cytometry replaces the normally-static sample plane of a microscope with a microfluidic device, enabling rapid image acquisition of many different objects. Thus far, these measurements have been limited to 1 and 2 dimensions. Here we discuss the implementation of advanced, 3D microscopy into an imaging flow cytometer and the unique calibration protocol we developed, in which we rely on statistical distributions rather than the unattainable static ground-truth. We demonstrate our system on live yeast cells, attaining 3D spatial information with orders of magnitude higher throughput than previous methods.