Comparison of conventional, confocal, and two-photon microscopy for detection of microcrystals within luminescence-based oxygen sensor films

Luminescence-based oxygen sensors, particularly those based on platinum-group complexes are of growing analytical importance. Commercial applications include aerodynamic studies of cars and aircraft in wind tunnels, monitoring of oxygen concentration during fermentation processes and in bioreactors, measurement of biological oxygen demand, and fluorescence detection and imaging of oxygen in blood, tissue, cells and other biological samples. Significant problems in the design and manufacture of polymer-supported, luminescence-based oxygen sensors include the observed non-linearity of the Stern-Volmer calibration plot and the multiexponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule within the polymer matrix. It will be shown that conventional, confocal, and two-photon fluorescence microscopy are invaluable tools with which microcrystals of the sensor molecule can be detected within sensor films. The design of the imaging systems, the measurement methods, and the results will be compared for the three approaches. As a result of the reduction in blur intensity and the minimization of photobleaching, two-photon microscopy provided the easiest and most effective method of microcrystal detection. The implications of the results in the rational design and mass production of luminescence-based oxygen sensors is discussed.