Many applications in chemistry, biology and medicine use microfluidic devices to separate, detect and analyze samples on a miniaturized size-level. Fluid flows evolving in channels of only several tens to hundreds of micrometers in size are often of a 3D nature, affecting the tailored transport of cells and particles. To analyze flow phenomena and local distributions of particles within those channels, astigmatic particle tracking velocimetry (APTV) has become a valuable tool, on condition that basic requirements like low optical aberrations and particles with a very narrow size distribution are fulfilled. Making use of the progress made in the field of machine vision, deep neural networks may help to overcome these limiting requirements, opening new fields of applications for APTV and allowing them to be used by nonexpert users. To qualify the use of a cascaded deep convolutional neural network (CNN) for particle detection and position regression, a detailed investigation was carried out starting from artificial particle images with known ground truth to real flow measurements inside a microchannel, using particles with uni- and bimodal size distributions. In the case of monodisperse particles, the mean absolute error and standard deviation of particle depth-position of less than and about 1 [my]m were determined, employing the deep neural network and the classical evaluation method based on the minimum Euclidean distance approach. While these values apply to all particle size distributions using the neural network, they continuously increase towards the margins of the measurement volume of about one order of magnitude for the classical method, if nonmonodisperse particles are used. Nevertheless, limiting the depth of measurement volume in between the two focal points of APTV, reliable flow measurements with low uncertainty are also possible with the classical evaluation method and polydisperse tracer particles. The results of the flow measurements presented herein confirm this finding. The source code of the deep neural network used here is available on https://github.com/SECSY-Group/DNN-APTV.