Despite the great potential of fluorescence microscopy, its application to date has largely been in the study of biological specimens. It will be shown that conventional fluorescence microscopy provides an invaluable tool with which to study the photophysics of polymer-supported luminescence-based oxygen sensors. The design of the imaging system, the measurement methods, and the data analysis used in the investigation of sensor systems are described. Fluorescence microscopic images of sensor films in which microheterogeneous regions exhibiting enhanced luminescence intensity and poorer oxygen quenching relative to the bulk response are shown. This is the first direct evidence that sensor molecules in various domains of the polymer support can exhibit different oxygen quenching properties. It will be shown that μ- and nano-crystallization of the sensor molecule are the probable source of both the observed heterogeneous microscopic responses and the microscopic and macroscopic nonlinear Stern-Volmer plots. The implications of these results in the rational design of luminescence-based oxygen sensors are discussed.