Fluorescence lifetime imaging is an optical imaging technique in which the brightness of each pixel represents fluorescence lifetime – rather than optical intensity. The fluorescence lifetime is a characteristic time during which a molecule remains in its excited state before emitting a photon. This characteristic time depends not only on the specific fluorophore but also on its environment. Molecular interactions influence relaxation processes modify fluorophore lifetimes. Therefore, FLIM can discriminate different stages of molecular interaction. Moreover, lifetime does not depend on molecular concentrations is therefore particularly beneficial for studying biochemical interactions on molecular scales where the fluorophore concentrations in a specimen are typically neither uniform nor steady.

In time-correlated single photon counting experiments like TCSPC-FLIM, the achievable time resolution is typically limited by the laser pulse duration the electronic jitter of the detector TDC. With the availability of ultrafast lasers, the pulse duration is not an issue for most experiments, while the detector's electronic jitter is often the key parameter. For instance, typical single-photon avalanche detectors (SPADs) have jitter on the order of 50 to 300 ps. On the other h , the development of modern superconducting nanowire single-photon detectors (SNSPDs) already provides a time resolution below to 15 picoseconds. To achieve the best possible time resolution with such detectors, it is desired that the TDC jitter is about a factor of two smaller than the detector jitter or lower.

Among various FLIM methods, the time-correlated single photon counting (TCSPC) approach delivers the highest time resolution photon detection efficiency. The figure above shows a typical setup that combines FLIM with scanning confocal microscopy to provide spatial filtering improved axial resolution. The essential ingredients are an x-y-z piezo scanner that scans the sample with a sub-micrometer resolution, a picosecond pulsed laser (Swabian Instruments DLnSec 520), a photon-counting detector (e.g., Excelitas AQRH). All signals are captured with a Swabian Instruments Time Tagger.

The system shown above provides great flexibility. It allows for on-the-fly, parallel processing storing of all signals. A set of powerful features makes sure that you minimize the time you spend on technical preparation system calibration. For example, you can easily compensate all cable delays with a precision of one picosecond directly in the software with one click. Virtual channels enable you to combine the counts from several detectors to represent total intensities or coincidence events between detectors.