Methods are provided for the automated detection, characterization, and selection of micro-objects in a microfluidic device. In addition, methods are provided for grouping detected micro-objects into subgroups that share the same characteristics and, optionally, repositioning micro-objects in a selected sub-population within the microfluidic device. For example, micro-objects in a selected sub-population can be moved into sequestration pens. The methods also provide for visual displays of the micro-object characteristics, such as two- or three-dimensional graphs, and for user-based definition and/or selection of sub-populations of the detected micro-objects. In addition, non-transitory computer-readable medium in which a program is stored and systems for carrying out any of the disclosed methods are provided.