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 and modify fluorophore lifetimes. Therefore, FLIM can discriminate different stages of molecular interaction. Moreover, lifetime does not depend on molecular concentrations and is therefore particularly beneficial for studying biochemical interactions on molecular scales where the fluorophore concentrations in a specimen are typically neither uniform nor steady.
Among various FLIM methods, the time-correlated single-photon counting (TCSPC) approach delivers the highest time resolution and photon detection efficiency. The figure above shows a typical setup that combines FLIM with scanning confocal microscopy, to provide spatial filtering and improved axial resolution. The essential ingredients are an x-y-z piezo scanner that scans the sample with sub-micrometer resolution, a picosecond pulsed laser (Swabian Instruments DLnSec 520), and a photon-counting detector (e.g. Excelitas AQRH). All signals are captured with a Swabian Instruments Time Tagger.
In time-correlated single-photon counting experiments like TCSPC-FLIM, the achievable time resolution is typically limited by the laser pulse duration and the electronic jitter of the detector and TDC. With the availability of ultrafast lasers, the pulse duration is not an issue for most experiments, while the electronic jitter of the detector 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 hand, the development of modern superconducting nano-wire 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.
The system shown above provides great flexibility. It allows for on-the-fly, parallel processing and storing of all signals. A set of powerful features makes sure that you minimize the time you spend on technical preparation and 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.
Your benefits from a Swabian Instruments FLIM system
You are ready for upcoming detector developments
The flexible input stages of Swabian Instruments Time Tagger Series allow you to seamlessly interface all common FLIM detectors with your system, including photo-multiplier-tubes (PMT), single-photon avalanche detectors (SPAD), and superconducting nanowire single-photon detectors (SNSPD), while taking advantage of the highest rise time of your signals. The high time resolution ensures that you are ready for new low jitter detectors that will be available in the future.
You are ready for multichannel FLIM and the implementation of your own novel imaging modes
Benefit from the high data rate and high channel count of Swabian’s Time Tagger to implement high-performance Fluorescence Lifetime Imaging experiments with multi-chromatic detection channels or novel imaging modes such as STED, PALM, and STORM By adding your own trigger signals, you can quickly develop your own novel imaging modes.
Automate your work – benefit from powerful native libraries in Matlab, Labview, Python, C#, C/C++
Swabian Instruments’ programming libraries enable you to implement a full-blown FLIM experiment with powerful lab automation capabilities within less than 10 lines of code (or less than 10 lab view VI’s) in your favorite programming language.