Fluorescence lifetime-based sensing and imaging

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics and chemical physics. However, time-resolved methods can also be used for chemical sensing. Lifetime-based sensing has several advantages over intensity-based methods. Since the lifetime is independent of the total probe intensity, its measurement can provide quantitative sensing of many analytes without the requirement for wavelength-ratiometric probes. Analytes like oxygen and halides can be determined by the collisional quenching mechanism. To date, lifetime probes for analyte recognition (binding) have been identified for Ca2+, Mg2+, K+ and pH. Importantly, the lifetime method provides a possibility to expand the sensitive analyte concentration range using probes with spectral shifts. The fluorescence lifetime method allows the sensing of analytes for which there are no direct probes, like glucose, antigens, or any affinity or immunoassays based on fluorescence energy transfer as the transduction mechanism. Advances in instrumentation, laser technology, fiber-optics and especially long-wavelength probes can result in the rapid migration of time-resolved fluorescence to clinical chemistry, environmental sensing and industrial applications. We shall describe phase-modulation instrumentation that can use simple light sources for which the light can be modulated externally by acoustooptic modulators or internally by driving current. Finally, we shall describe fluorescence lifetime imaging microscopy (FLIM), in which image contrast is created from the lifetime at each point of the image. Time-resolved imaging is now a reality in fluorescence microscopy, and promises to provide chemical imaging of a variety of intracellular analyte and/or cellular phenomena.