Super-resolution imaging using fluorescence lifetimes and phasor analysis

Fluorescence microscopy is an important tool in many scientific areas. The development of fluorescence super-resolution microscopy has overcome the traditional limitation of spatial resolution to half the wavelength of light, enabling the imaging of biological macromolecules at the nanometer scale. Here, we discuss how the analysis of fluorescence lifetimes and the phasor analysis can be used as an original strategy to perform super-resolution imaging. Specifically we describe a method developed in the context of stimulated emission depletion (STED) microscopy called separation of photons by lifetime tuning (SPLIT) and its different implementations. To quantify the improvement of resolution provided by SPLIT we use a statistical method that evaluates the image quality by image correlation spectroscopy (QuICS). As an example of application, we show SPLIT images of transcription foci in U937-PR9 cells and evaluate the improvement of resolution provided by SPLIT compared to conventional STED.