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 stream ¹. 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 second ². 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 distinguishes cells in the flow by measuring differences in fluorescence intensity or by separating fluorophores based on their emission spectra. This works well as long as each fluorophore’s bandwidth can be distinguished from others, or the brightness difference between them is resolvable. When signals are too weak or exhibit rapid variations, this method begins to show its limitations.

Fluorescence lifetime flow cytometry (FLFC), also known as photon counting flow cytometry (PCFC), overcomes key limitations of conventional flow cytometry by introducing fluorescence lifetime as an additional parameter. This enables robust discrimination of fluorophores even when their emission spectra overlap. Fluorescence lifetimes are measured using pulsed laser excitation in combination with single-photon detectors (SPD), typically implemented via time-correlated single-photon counting (TCSPC). This approach captures the intrinsic photon statistics of individual fluorophores in the time domain, providing a powerful contrast mechanism beyond intensity and spectral information. By exploiting differences in fluorescence decay dynamics, FLFC improves fluorophore discrimination under challenging conditions, including low signal levels or strong spectral overlap. Lifetime information can be extracted using decay fitting, curve-shape analysis, or phasor analysis. The resulting time-resolved datasets enhance overall data quality and enable model-based analyses of fluorescence dynamics at the single-molecule level, substantially expanding the diagnostic and research capabilities of flow cytometry ⁵. When combined with simple spectral gating, FLFC can also resolve weak, broadband autofluorescence from endogenous metabolic cofactors, enabling fully label-free single-cell phenotyping 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 stream ¹. Samples may be analyzed 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, FLFC can provide improved SNR in low-light regimes compared to 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 fluorescent lifetimes, while still enabling label-free analysis of cells via autofluorescence. Despite these advantages, FLFC setups still face some intrinsic limitations, including:

• Signal Synchronization: FLFC setups can scale up with multiple pulsed lasers and detectors. Measuring, synchronizing, and analysing all these signals can be challenging for conventional timing electronics.

• Real-Time Analysis: FLFC is typically slower than conventional flow cytometry due to limited photon statistics per cell and the computational overhead of fluorescence lifetime estimation and downstream decision-making. These constraints complicate real-time analysis at high flow rates.

• Post-Processing Analysis: Post-processing FLFC data is challenging due to large time-tagged datasets. Photon events must be correctly assigned to individual cells and excitation cycles. Lifetime extraction and subsequent statistical analysis are sensitive to model assumptions, affecting robustness and reproducibility.

• Cell-synchronous measurements: While cell presence in FLFC is commonly detected using forward- or side-scattering signals, this information is often processed outside the time-resolved measurement chain. As a result, acquisition parameters are typically defined conservatively, limiting adaptive measurements triggered by each cell and reducing overall efficiency.

Swabian Instruments’ Time Taggers offer a versatile tool for FLFC experiments, particularly due to the following acquisition and analysis features:

• Trigger Handling and Synchronization: Swabian Instruments’ Time Taggers provide full timestamping and deterministic synchronization of laser trigger signals and photon arrival times at repetition rates up to several tens of MHz. This enables high-fidelity fluorescence lifetime histogramming with well-defined temporal alignment across excitation and detection channels, even when scaled up to multiple lasers and detectors.

• Real-Time Analysis: Time Taggers from Swabian Instruments support on-the-fly computation of fluorescence lifetime histograms and real-time phasor analysis. Extracted lifetime information is streamed continuously via USB to the host system, enabling immediate downstream processing, classification, or control logic during acquisition.

• Post-Processing Analysis: Swabian Instruments’ Time Taggers record complete time-tag streams, allowing experiments to be replayed and re-analyzed at any time. This enables lifetime extraction and data analysis with modified parameters or alternative models, even after the sample or measurement conditions are no longer available.

• High-Speed Custom Measurements: The Time Tagger platform allows users to implement custom, hardware-level analysis logic that operates directly on the time-tag stream in real time. This enables application-specific measurement strategies with full flexibility. For example, the Melissa Skala Lab demonstrated an increase in FLFC throughput by implementing custom logic that replaces side-scatter–based cell detection with time-resolved photon analysis. ⁹

Overall, Swabian Instruments’ Time Taggers are particularly valuable for time-resolved photon counting and, therefore, 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 full flexibility and full access to the photon arrival distribution. Swabian Instruments’ Time Taggers have a proven record of success in multi-channel, high-precision, time-stamped photon counting applications ^{5 6}. Implementing Swabian Instruments’ Time Taggers into FLFC experiments allows workflows to be more accessible, scalable, and reproducible. 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) and pushed FLFC to record-speed, using Swabian Instruments’ Custom Measurement ability ¹⁰.

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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