Fluorescence Lifetime Imaging (FLIM)

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 Tagger 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 Tagger 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 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 Tagger allows the precise determination of photons’ arrival time with a timing jitter down to 1.5 ps.

The critical hardware parameters necessary for FLIM applications are:

• Timing jitter. The lower the timing jitter of the timing electronics is, the better the resolution and precision of the measurement will be. One has to take into account the jitter from the detector and try to keep the timing jitter from the electronics below this value.
• Multi-channel configuration. Acquiring information from the light source, the detector, and the piezo stage simultaneously requires electronics capable of acquiring and precisely correlating multiple signals. This becomes essential when the experiment involves associating detector signals with a time-window for each given pixel. The complexity scales up further when the experiment demands high spatial resolution.
• Data transfer rate. Higher data transfer rates allow the capture of more photon events in a shorter time window, thereby increasing the statistical significance of measurements. Insufficient data transfer leads to overflow and data loss unless data filtering methods exist to control data acquisition before being transferred to the PC. The successful data filtering method will utilize the total sum of the count rates for each of the channels (including laser, detector, controllers, signal triggers, etc) to determine the clicks that must be transferred.

Time-Correlated Single-Photon Counting with Single Quantum EOS SNSPD System

TCSPC_Swabian_Instruments.pdf

Photon Number Resolution with Room Temperature Detector

Photon_number_resolution_with_room_temperature_detector.pdf

Customer success stories

January 22, 2025

Simplifying Photon Detection and Analysis with Time Taggers

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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