Fluorescence Lifetime Flow Cytometry (FLFC)

Flow cytometry is a sheath-flow-based technique that uses laser excitation and fluorescence to evaluate the physical and chemical characteristics of cells or other suspended particles in a fluid stream¹. As these particles pass through the laser beam, the scattered light and fluorescence from labeled antibodies or markers can be used to obtain information on size, granularity, and molecular expression at the single-cell level, at rates exceeding tens of thousands of cells per second². Flow cytometry is widely used across biomedical research and clinical diagnostics, including oncology, immunology, stem cell studies, microbiology, and synthetic biology. It offers high-throughput quantitative analysis of heterogeneous cell populations, which is crucial in a variety of applications such as cancer phenotyping, vaccine development, and rapid diagnosis of blood cancers or infectious diseases^{3 4}.

Conventional flow cytometry measures fluorescence by integrating the analog detector signal over time, a process that often obscures subtle temporal fluorescence variations, especially when signals are weak or rapidly changing. As modern systems push for higher speed, multiplexing, and sensitivity, these limitations become more pronounced. To meet those demands, Fluorescence Lifetime Flow Cytometry (FLFC), also known as Photon Counting Flow Cytometry (PCFC), precisely captures the arrival times of individual photons emitted by fluorophores - either external labels or intrinsic metabolites - within cells or other suspended particles, revealing dynamic fluorescence information.

Single-photon counting (SPC) is particularly helpful when fluorescence signals are intrinsically weak; for example, when probing low-abundance targets, using dim fluorophores, or analysing intrinsic cellular autofluorescence. Because the technique of SPC counts individual quanta, the noise floor is limited only by shot noise (Poisson statistics), and it avoids the additional electronic noise introduced when analog signals are integrated. Moreover, photon-counting flow cytometry improves fluorophore discrimination by exploiting differences in fluorescence-decay time, even when signals are weak or emission spectra overlap. The differences can be detected either via lifetime fitting, curve-shape analysis, or phasor analysis. The resulting time-resolved dataset enhances overall data quality and supports model-based analyses of fluorescence dynamics at the single-molecule level, greatly extending the diagnostic and research capabilities of flow cytometry⁵. Coupled with simple spectral gating, it can also resolve the weak, broadband autofluorescence of key metabolic cofactors, enabling fully label-free phenotyping at the single-cell level and revealing changes in cellular metabolism^{6 7}.

A typical Fluorescence Lifetime Flow Cytometry (FLFC) system begins with a sheath-flow fluidics unit that aligns cells in a single, gentle stream¹. Samples may be analysed label-free via intrinsic autofluorescence or, as in standard flow cytometry, after labelling with specific fluorescent probes^{2 8}. The optical path comprises one or more excitation lasers, dichroic mirrors, and band-pass filters, followed by single-photon detectors such as PhotoMultiplier Tubes (PMTs) or Avalanche Photo Diodes (APDs/SPADs).

In some setups, a signal generator can be introduced to orchestrate the experiment and dictate the timing of the laser excitation pulses. The laser output acts as the start signal, and each detected photon acts as the stop signal. High-precision timing electronics, such as Swabian Instruments’ Time Taggers, record both start and stop signals with picosecond-level precision, allowing per-cell fluorescence-lifetime calculation. After optical interrogation, the stream can feed a sorting module for real-time decision-making regarding where to direct the stream of cells; for example, it can be based on whether a cell contains a certain fluorophore or another.

From a technical standpoint, Fluorescence Lifetime Flow Cytometry (FLFC) delivers a higher SNR than conventional Flow Cytometry because every photon is counted individually, and there is no analog baseline to correct. It distinguishes dyes with overlapping emission spectra by using their different decay times while still analysing label-free cells via autofluorescence and requiring fewer detector channels than a purely spectral setup. Despite these advantages, there are intrinsic common limitations in FLFC setups, including:

• Limited time resolution: Fixed-window analog integration discards per-photon arrival times (e.g., microtime/lifetime data), limiting analysis of rapid dynamics, photon-arrival statistics, and transient events. This results in a limited amount of information that can be captured from each cell or particle under study. As a consequence, separating fluorophores with similar emission spectra and quantifying weak signals in multiplexed panels becomes significantly more difficult⁵.

• High timing jitter negatively affects the precision of lifetime and time-of-flight measurements, resulting in a decrease in the accuracy of fluorophores’ classification.

• Long dead time: After each detection, the electronics remain insensitive for a finite interval, causing event losses and pile-up bias at high photon rates. Consequences include undercounted intensity, distorted lifetime histograms (early photons over-represented), reduced dynamic range, and lower maximum throughput per channel.

• Synchronization complexity: Aligning the laser timing reference, detector channels, and the cell-presence trigger (e.g., side scatter) typically requires external fan-out or delay hardware. Possible misalignment leads to incorrect per-cell assignment and biased lifetime estimates.

• Low flexibility in the acquisition and analysis: A reduced number of input channels that are fixed to the conventional start/stop architecture restricts simultaneous recording of multiple detectors and triggers. Fixed or non-programmable settings (such as fixed bin widths, ranges, and gating) limit the capabilities of lifetime characterization via multi-exponential fits and phasor approaches. This also results in constrained real-time tasks such as adaptive gating or sorting, reducing the scope for post-hoc reanalysis and system scalability.

Swabian Instruments’ Time Taggers offer a versatile tool for Fluorescence Lifetime Flow Cytometry (FLFC) experiments, particularly due to the following acquisition and analysis features:

• High Input frequency: Full timestamping of the laser pulse and photon arrival with frequencies up to several hundred MHz results in precise lifetime histograms for sample characterization.

• Picosecond jitter: Swabian Instruments’ Time Taggers can achieve timing jitter down to 1.5 ps root-mean-square (RMS), enabling highly accurate measurements when paired with single-photon detectors for FLFC. This ultra-low jitter ensures precise timing, enhancing sensitivity to weak (auto)fluorescent signals and enabling detailed analysis at very low light conditions.

• No dead time between detector channels (intrachannel dead time ≤ 6 ns, model-dependent).

• Synchronized measurements achieved by combining Pulse Streamer 8/2 (a signal generator with 8 digital outputs, 1 GSa/s, and 2 ns pulse width for laser triggering and setup orchestration) with Time Taggers, due to the integrated trigger handling and synchronization capabilities leveraging its built-in internal oscillator. In short, the Time Tagger acquires signals from photon detectors (e.g., PMTs, SPADs) and coordinates with excitation laser pulses or sample triggers, which can be precisely defined with the Pulse Streamer 8/2.

• From an analysis perspective, Swabian Instruments’ Time Taggers can compute fluorescence lifetime histograms on the fly, allow phasor analysis on the run or during post-analysis, and, for example, real-time sorting or merging/discarding of multi-channel histogram data to gather cleaner data for analysis.

Overall, Swabian Instruments’ Time Taggers are particularly valuable for time-resolved photon counting and, therefore, Fluorescence Lifetime Flow Cytometry (FLFC) setups due to their unique combination of powerful hardware capabilities and software-defined approach to data analysis. Integrating Swabian Instruments Time Taggers into these experiments results in precise measurements with picosecond-jitter that allow for detailed analysis of photon arrival distribution, intensity, and duration per event. Swabian Instruments’ Time Taggers have a proven record of success in multi-channel, high-precision, time-stamped photon counting applications [Samimi references]. Implementing Swabian Instruments’ Time Taggers into advanced Fluorescence Lifetime Flow Cytometry (FLFC) experiments allows workflows to be more accessible, scalable, and reproducible. By adding precise photon counting capabilities and fluorescence lifetime analysis, flow cytometry moves beyond counting fluorophores expression-level (intensity) to measuring the functioning of proteins within the cell environment. This opens new frontiers in metabolism, signaling, and immunophenotyping ⁷. This is particularly valuable for interdisciplinary teams building custom flow cytometers, such as Prof. Melissa Skala’s group, a renowned bioimaging scientist. Skala’s group has extensive experience in developing time-resolved single-photon detection systems for flow cytometry, fluorescence lifetime analysis, and single-molecule detection applications (documentation in this SkalaLab Github repository ⁹).

October 7, 2025

Multichannel Continuous Histogramming: Advancing Fluorescence Lifetime Flow Cytometry with Melissa Skala’s Team and Swabian Instruments

Discover Swabian Instruments Time Tagger’s capability for multi-channel continuous histogramming, which has resulted in further advancements in the cutting-edge work of Prof. Melissa Skala’s group at the Morgridge Institute for Research and the University of Wisconsin-Madison.

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