Fluorescence lifetime imaging microscopy (FLIM)

Fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging technique that reveals both the structure and dynamic behavior of fluorescent samples. This powerful method combines high-resolution microscopy with precise measurements of fluorescence decay times, offering unique insights into cellular and molecular processes.

Many species such as organic fluorophores, fluorescent proteins, or nanoparticles (quantum dots and nanodiamonds) can emit photons after absorbing light in a process called fluorescence. The fluorescence lifetime is an intrinsic characteristic of such a light emitting species and indicates how long it remains in an electronically excited state before returning to the ground state. Each emitting species has a characteristic fluorescence lifetime that can be influenced by its environment.

FLIM produces an image based on the differences in excited state decay rates. Thus, FLIM is an imaging technique where the contrast is based on the lifetimes of individual fluorophores rather than their emission spectra.

SPAD arrays eliminate pile-up, a limitation requiring conventional FLIM detectors to have a 10% detection rate with respect to the laser repetition rate. Due to conventional instrumentation dead time, early photons are more likely to be detected and late photons are ignored, leading to a systematic error. SPAD arrays push this limitation by spreading photons to multiple pixels, thus allowing for more likely detection of late photons.