Conventional, confocal and two-photon fluorescence microscopy investigations of polymer-supported oxygen sensors

Luminescence-based, polymer-supported oxygen sensors, particularly those based on platinum group complexes, continue to be of analytical importance. Commercial applications range from the macroscopic (e.g. aerodynamic investigations in wind tunnels, monitoring of oxygen concentration during fermentation, and measurement of biological oxygen demand) to the microscopic (e.g. imaging of oxygen in blood, tissue, cells and other biological samples). Problems hindering the design of improved oxygen sensors include non-linear Stern-Volmer calibration plots and the multi-exponentiality of measured lifetime decays, both of which are attributed primarily to heterogeneity of the sensor molecule in the polymer support matrix. Conventional, confocal and two-photon fluorescence microscopy have proven to be invaluable tools with which the microscale heterogeneity and response of luminescence-based oxygen sensors can be investigated and compared to the macroscopic response. Results obtained for three ruthenium(II) alpha-diimine complexes in polydimethylsiloxane polymer supports indicate the presence of unquenched microcrystals within the polymer matrix that probably degrade oxygen quenching sensitivity and linearity of the Stern-Volmer quenching plot. Two-photon fluorescence microscopy proved most useful for imaging microcrystals within sensor films, and conventional microscopy allowed direct comparison between microscopic and macroscopic sensor response. The implications of the results in the rational design and mass production of luminescence-based oxygen sensors are significant.