Overview of the technique
Observing the environment of molecules through imaging is critical to understanding their chemical behaviour. Intensity based methods alone are not fully capable of providing this information. Excited state measurements are arguably the only and best method to achieve this. Lifetime imaging microscopy is a technique where the luminescence (fluorescence or phosphorescence) lifetime variation across the sample creates the contrast in the image. The lifetime may be determined by frequency modulation, camera-based time gating or using time-correlated single photon counting (TCSPC).
This latter method gives the best spatial resolution when combined with multiphoton and confocal as well as time resolution (picosecond). Electrons in the molecule are excited with an ultra-short, pulsed laser (to match the absorption) and the average decay process recorded from each pixel is determined by the TCSPC technique together with an equally very fast point detectors all synchronised together to give this technique its advantage.
Fluorescence and phosphorescence lifetime imaging microscopies (FLIM and PLIM) have found applications at the interface between life and physical sciences with relevance to biomedicine. Both FLIM and PLIM can elucidate the reaction process and subsequent fate of drugs and ordinary imaging probes located within specific cellular organelle and environment.
We employ the FLIM or PLIM technique which simultaneously records both in confocal or multiphoton. FLIM provides information of the molecule during a singlet-singlet electronic transition whilst PLIM gives information about the forbidden singlet-triplet processes. These are instantaneously recorded on a pixel-by-pixel basis (300nm spatial resolution) and a time range of picoseconds to milliseconds. PLIM is particularly suited for the study of molecules that are long-lived in their excited states.