Figure 1. Conceptual scheme of the physics behind the fluorescence lifetime imaging (FLIM). (a) Illustration of the molecular excitation and relaxation process. Upon photon absorption, an electron in the molecule makes a transition from the ground state to an excited state. After undergoing vibrational relaxation, the electron returns to the ground state. During this transition, the release of a photon with lower energy and a characteristic lifetime occurs. (b) The plot depicts the photon excitation (blue curve) and the exponential decrease in fluorescence intensity (red curve), highlighting the characteristic lifetime, which reflects molecular properties and the surrounding environment.
Fluorescence lifetime imaging (FLIM) is an imaging technique for mapping the spatial distribution of fluorescence lifetime within a sample. Unlike traditional imaging techniques, where each pixel corresponds to optical intensity, FLIM associates each pixel with the fluorescence lifetime of the molecule. The fluorescence lifetime is the characteristic time during which a fluorescent molecule (fluorophore) remains in the excited state before returning to the ground state and emitting a photon.
Figure 1 depicts the basic concept behind FLIM. When a photon impinges on a molecule, an electron can be promoted from the ground state to an excited state. Within the excited electronic state, the electron can relax to lower vibrational sublevels through vibrational relaxation, dissipating energy due to interactions with the molecule's surroundings (e.g., other molecules or solvents). After this process, it decays to the ground state, resulting in the emission of a photon after a characteristic delay known as the fluorescence lifetime.
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 be used to discriminate different stages of molecular interaction. Additionally, because fluorescence lifetime is independent of molecular concentration, FLIM is particularly advantageous for studying biochemical interactions at molecular scales, where fluorophore concentrations are often heterogeneous or variable.
Among various FLIM methods, the time-correlated single photon counting (TCSPC) approach delivers the highest time resolution and photon detection efficiency. Swabian Instruments’ Time Taggers are specifically designed to address the requirements of such high-precision measurements. Interfacing with a wide range of photon detectors - including photo-multiplier-tubes (PMT), single-photon avalanche detectors (SPAD), and superconducting nanowire single-photon detectors (SNSPD) - the Time Taggers can measure the time difference between excitation photons and those emitted during fluorescence decays, thereby enabling the precise evaluation of the fluorescence lifetime of the sample.
This technique can be applied to measure not only a single point but also to scan the whole sample. Employing advanced methodologies such as 3D piezo scanning or light steering via digital micromirror devices, one can obtain an image with a full map of fluorescence lifetimes. These approaches provide a comprehensive view of molecular interactions and their dynamic processes.
The FLIM technique has various applications for investigating biological, chemical, and physical processes at the microscopic scale. To mention a few: interactions between proteins using FRET-FLIM [1], and studies on cancer cell’s microenvironments [2].
Figure 2. Schematical representation of a typical setup that combines Fluorescence lifetime imaging (FLIM) with scanning confocal microscopy.
Figure 2 shows a typical setup for fluorescence lifetime imaging (FLIM) measurements. In this example, the setup employs scanning confocal microscopy, a widely used implementation for FLIM experiments. Photons are sent from a light source - typically a picosecond pulsed laser - directly onto the sample through a microscope. This optical system is used to reduce the beam spot size, enabling sub-micrometer precision and high resolution. The fluorescence photons produced are then sent to a single-photon detector. The acquisition is correlated with the corresponding sample position, which is precisely controlled by moving the sample with a 3D piezo stage. All of the signals - laser-clicks, photons-clicks, and time-tags associated with pixel position - are acquired simultaneously and in real-time by the Time Tagger.
In FLIM the achievable time resolution is typically limited by the laser pulse duration and the electronic jitter of the detector and time-to-digital converter (TDC). With the availability of ultrafast lasers, the laser pulse duration is no longer a limiting factor. At the same time, the jitter from detectors and/or the read-out electronics often becomes the key parameter to consider. For instance, typical single-photon avalanche detectors (SPADs) have jitter on the order of tens to hundreds of picoseconds. In contrast, modern superconducting nanowire single-photon detectors (SNSPDs) achieve time resolutions below 10 picoseconds. To fully exploit the capabilities of these detectors, the timing electronics must have jitter below the one of the detector. Swabian Instruments' Time Taggers allows the precise determination of photons' arrival time with a timing jitter down to 1.3 ps.
The critical hardware parameters necessary for FLIM applications are:
In fluorescence lifetime imaging (FLIM), it is important to acquire different signals simultaneously and correlate them with high precision. Swabian Instruments' software offers a user-friendly, easily implementable solution that supports real-time measurements. The software’s features, such as picosecond-precision delay compensation and virtual channels, significantly reduce setup time and streamline calibration. Virtual channels, in particular, enable simultaneous tracking of multiple photon detection events, making them particularly valuable for experiments involving intensity correlation analysis or coincidence counting. As an example, the time tags acquired from the piezo-scanner allow the definition of precise time gates, enabling photons to be accurately mapped to their corresponding pixels. The Swabian Instruments documentation provides comprehensive guidance for configuring these features, along with numerous other advanced applications.
Swabian Instruments' programming libraries enable you to implement a full-blown FLIM experiment with powerful lab automation capabilities within just a few lines of code in your favorite programming language.
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). The high time resolution ensures that you are ready for new low jitter detectors that will be available in the future.
Benefit from the high data rate and high channel count of Swabian Instruments’ Time Tagger to implement high-performance FLIM experiments with multi-chromatic detection channels or novel imaging modes. By adding your own trigger signals, you can quickly develop your own novel imaging modes.
[1] Gadella, T. W., & Jovin, T. M., „Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy." Journal of Cell Biology, 129(6), 1543-1558 (1995).
[2] Lakowicz, J. R., et al., „Fluorescence lifetime imaging of free and protein-bound NADH.", Proceedings of the National Academy of Sciences, 89(4), 1271-1275 (1992).
Swabian's Time Tagger Series enables you to capture not only all your single photons but also all your other signals such as triggers from microwave sources, fast switches, pulse pattern generators, piezo-scanners, etc. to implement the entire data acquisition for your quantum optics experiment with single hardware with only few lines of code.
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Photon detection distinguishability is increasing interest in advancements in photonics and quantum technology. At Swabian Instruments, we are proud to lead this innovation with our Time Taggers , as detailed in our latest abstract presented at the Optica Latin America Optics and Photonics Conference, which took place in Puerto Vallarta, Mexico, in November 2024.
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